Bioluminescence: Methods and Protocols [2 ed.] 9781603273206, 1603273204

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METHODS

IN

M O L E C U L A R B I O L O G Y TM

Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK

For other titles published in this series, go to www.springer.com/series/7651

Bioluminescence Methods and Protocols Second Edition

Edited by

Preston B. Rich and

Christelle Douillet University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

Editors Preston B. Rich Department of Surgery University of North Carolina Division of Trauma & Critical Care 4008 Burnett-Womack Bldg. Chapel Hill, NC 27599-7228 USA [email protected]

Christelle Douillet Department of Surgery University of North Carolina Division of Trauma & Critical Care 90 Manning Drive 6119A Thurston-Bowles Bldg. CB# 7161 Chapel Hill, NC 27599-7161 USA [email protected]

Series Editor John M. Walker University of Hertfordshire Hatfield, Herts UK

ISSN 1064-3745 e-ISSN 1940-6029 ISBN 978-1-60327-320-6 e-ISBN 978-1-60327-321-3 DOI 10.1007/978-1-60327-321-3 Springer Dordrecht Heidelberg London New York Library of Congress Control Number: 2009931798 # Humana Press, a part of Springer ScienceþBusiness Media, LLC 2009 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer ScienceþBusiness Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper Springer is part of Springer ScienceþBusiness Media (www.springer.com)

Preface Part I I was raised in a redbrick Baltimore row house where summer was marked by the timehonored ritual of firefly-chasing – a backyard tradition that has endured the generations. Amid the excitement, my father often told the story of how, when he was a child, researchers at the Johns Hopkins University had appealed for the systematic capture of live fireflies en masse. Science had engaged the Baltimore youth in an entrepreneurial quest to jar as many lightning bugs as the dwindling light of dusk would permit. The very next morning, each 100-count glass jar of glowing crawling insects could be exchanged at the University for exactly one crisp dollar bill. Unrecognized at the time by my father, his joyous endeavors had contributed in a profound way to the advanced molecular biological techniques that serve as the basis for this textbook. In 1947, William McElroy used extracts from those very fireflies to define the fundamental reaction underlying the mystical phenomenon of luminescence, and published ‘‘The energy source for bioluminescence in an isolated system’’ in the Proceedings of the National Academy of Sciences. In the decades since that summer, the study and application of bioluminescence have allowed us to leverage the enduring power of nature’s elegance. We have painstakingly harnessed a powerful tool that enables us to seek a deeper understanding of the complex mechanisms underpinning so many vital biologic systems. This second edition of Methods in Molecular Biology’s Bioluminescence: Methods and Protocols serves as a readable and utilitarian compilation of the newest and most innovative techniques that have emerged in this rapidly expanding and progressively diverse field. We are indebted to the authors for their thoughtful contributions, inspired by their rigorous dedication to the science of bioluminescence, humbled by the unyielding support of our colleagues, and grateful for the opportunity provided us by John and Jan Walker. Chapel Hill, North Carolina

Preston B. Rich

Part II My first encounter with bioluminescence happened while I was a child, during a family vacation at an Atlantic beach. One evening, we stayed near the water until night time, seeking some relief from the unusual heat. The magic happened when each walking and kicking step agitated the sand and water, lighting a soft blue glow. It evoked both poetic wonder and the foreign feeling suggested by a sci-fi movie. Later, I speculated which species was present that day. It is difficult to know, since so many marine organisms are bioluminescent. v

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Our personal anecdotes and countless others illustrate the widespread occurrence of bioluminescence in nature (bacteria, fungi, worms, fireflies, coral, jellyfish, fishes, etc.). Indeed, it appeared many times independently throughout evolution. Its purposes are also varied: it can be used for communication, predation (e.g., attraction to a lure in fish or aggressive flashing mimicry in fireflies), reproduction (attracting a mate), camouflage, repulsion, or other defensive strategies (e.g., dinoflagellates when endangered by a predator may use bioluminescence to attract a bigger predator who may prey on the smaller predator), and sometimes for illumination (night vision). The extensive use of bioluminescence in nature is mirrored by its very wide use in scientific laboratories. Since the classical experiment by Raphael Dubois in 1885 describing for the first time the luciferin–luciferase reaction, the applications of bioengineered bioluminescence have continuously increased in number. In popular culture, the development of glowing pets, self-illuminating Christmas trees, and other wild endeavors appear amusing (light indeed). However, the applications in biotechnology and medicine are cutting-edge and far-reaching. Bioluminescence is used to study cellular and subcellular phenomenon, and we present in this second edition of Methods in Molecular Biology’s Bioluminescence: Methods and Protocols some methods to assess cell trafficking, protein–protein interactions, intracellular signaling, and apoptosis. One key feature of bioluminescence is the possibility to visualize and quantify biological mechanisms in real-time and in in vivo settings. This opens new avenues of knowledge, and we have included here some chapters that describe the in vivo study of bacterial or viral infections, transplanted cells, stem cells proliferation, vascular flow, and tumors. The commercialization of reporter genes, assay kits, and imaging systems provide easy access to the materials needed for such studies. This book provides protocols that are detailed enough to be followed and adapted by scientific teams who have no previous expertise in bioluminescence. Hence, we believe that numerous breakthrough and new applications from basic to applied science and medicine will continue to be developed. We thank the chapters’ authors for sharing their rich expertise, and Jan and John Walker for helping us throughout the editorial process. Chapel Hill, North Carolina

Christelle Douillet

Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1.

Luminescent Probes and Visualization of Bioluminescence . . . . . . . . . . . . . . . . . . Elisa Michelini, Luca Cevenini, Laura Mezzanotte, and Aldo Roda

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2.

Validation of Bioluminescent Imaging Techniques. . . . . . . . . . . . . . . . . . . . . . . . . John Virostko and E. Duco Jansen

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Assessment of Extracellular ATP Concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . Lucia Seminario-Vidal, Eduardo R. Lazarowski, and Seiko F. Okada

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High-Throughput Quantitative Bioluminescence Imaging for Assessing Tumor Burden . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Angelina Contero, Edmond Richer, Ana Gondim, and Ralph P. Mason

5.

6.

Fluorescence Imaging of Tumors with ‘‘Smart’’ pH-Activatable Targeted Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Daisuke Asanuma, Hisataka Kobayashi, Tetsuo Nagano, and Yasuteru Urano Imaging Vasculature and Lymphatic Flow in Mice Using Quantum Dots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Byron Ballou, Lauren A. Ernst, Susan Andreko, James A. J. Fitzpatrick, B. Christoffer Lagerholm, Alan S. Waggoner, and Marcel P. Bruchez

7.

Bioluminescent Imaging of Transplanted Islets . . . . . . . . . . . . . . . . . . . . . . . . . . . Xiaojuan Chen and Dixon B. Kaufman

8.

Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cell Survival, Proliferation, and Fate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Kitchener D. Wilson, Mei Huang, and Joseph C. Wu

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Detection of Apoptosis Using Cyclic Luciferase in Living Mammals . . . . . . . . . . . 105 Akira Kanno, Yoshio Umezawa, and Takeaki Ozawa

10. Noninvasive Bioluminescent Imaging of Infections . . . . . . . . . . . . . . . . . . . . . . . . 115 Javier S. Burgos 11. Real-Time Bioluminescence Imaging of Viral Pathogenesis . . . . . . . . . . . . . . . . . . 125 Kathryn E. Luker and Gary D. Luker 12. Bioluminescent Monitoring of In Vivo Colonization and Clearance Dynamics by Light-Emitting Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137 Siouxsie Wiles, Brian D. Robertson, Gad Frankel, and Angela Kerton 13. Quantitative In Vivo Imaging of Non-viral-Mediated Gene Expression and RNAi-Mediated Knockdown . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155 Garrett R. Rettig and Kevin G. Rice

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14. Analysis of Protein–Protein Interactions Using Bioluminescence Resonance Energy Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 Kevin D.G. Pfleger 15. Bioluminescent Imaging of MAPK Function with Intein-Mediated Reporter Gene Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185 Akira Kanno, Takeaki Ozawa, and Yoshio Umezawa 16. Bioluminescence Analysis of Smad-Dependent TGF-b Signaling in Live Mice . . . . 193 Jian Luo and Tony Wyss-Coray 17. Bioluminescence Imaging of Calcium Oscillations Inside Intracellular Organelles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203 Carlos Villalobos, Marı´a Teresa Alonso, and Javier Garcı´a-Sancho 18. Novel Tools for Use in Bioluminescence Resonance Energy Transfer (BRET) Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215 Me´lanie Robitaille, Isabelle He´roux, Alessandra Baragli, and Terence E. He´bert 19. PIN-G Reporter for Imaging and Defining Trafficking Signals in Membrane Proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235 Lynn Mckeown, Vicky C. Jones, and Owen T. Jones 20. Imaging b-Galactosidase Activity In Vivo Using Sequential Reporter-Enzyme Luminescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249 Georges von Degenfeld, Tom S. Wehrman, and Helen M. Blau Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261

Contributors MARI´A TERESA ALONSO • Instituto de Biologı´a y Gene´tica Molecular (IBGM), Universidad de Valladolid and Consejo Superior de Investigaciones Cientı´ficas (CSIC), Valladolid, Spain SUSAN ANDREKO • Molecular Biosensor and Imaging Center, Carnegie Mellon University, Pittsburgh, PA, USA DAISUKE ASANUMA • Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan BYRON BALLOU • Molecular Biosensor and Imaging Center, Carnegie Mellon University, Pittsburgh, PA, USA ALESSANDRA BARAGLI • Department of Pharmacology and Therapeutics, McGill University, Montre´al, QC, Canada HELEN M. BLAU • Baxter Laboratory in Genetic Pharmacology, Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA MARCEL P. BRUCHEZ • Molecular Biosensor and Imaging Center and Department of Chemistry, Carnegie Mellon University, Pittsburgh, PA, USA JAVIER S. BURGOS • Drug Discovery Unit, Neuron BPh, Granada, Spain LUCA CEVENINI • Department of Pharmaceutical Sciences, University of Bologna, Bologna, Italy ANGELINA CONTERO • Cancer Imaging Program, UT Southwestern, Dallas, TX, USA XIAOJUAN CHEN • Division of Organ Transplantation, Department of Surgery, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA E. DUCO • Jansen Department of Biomedical Engineering, Vanderbilt University, Nashville, TN, USA LAUREN A. ERNST • Molecular Biosensor and Imaging Center, Carnegie Mellon University, Pittsburgh, PA, USA JAMES A.J. FITZPATRICK • Molecular Biosensor and Imaging Center, Carnegie Mellon University, Pittsburgh, PA, USA GAD FRANKEL • Division of Cell and Molecular Biology, Imperial College, London, UK JAVIER GARCI´A-SANCHO • Instituto de Biologı´a y Gene´tica Molecular (IBGM), Universidad de Valladolid and Consejo Superior de Investigaciones Cientı´ficas (CSIC), Valladolid, Spain ANA GONDIM • Cancer Imaging Program, UT Southwestern, Dallas, TX, USA TERENCE E. HE´BERT • Department of Pharmacology and Therapeutics, McGill University, Montre´al, QC, Canada ISABELLE HE´ROUX • De´partement de Biochimie, Universite´ de Montre´al; Department of Pharmacology and Therapeutics, McGill University, Montre´al, QC, Canada MEI HUANG • Molecular Imaging Program at Stanford (MIPS), Stanford University, Stanford, CA, USA OWEN T. JONES • Faculty of Life Sciences, The University of Manchester, Manchester, UK VICKY C. JONES • Faculty of Life Sciences, The University of Manchester, Manchester, UK ix

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AKIRA KANNO • Department of Chemistry, School of Science, The University of Tokyo; Japan Science and Technology Agency, Tokyo, Japan DIXON B. KAUFMAN • Division of Organ Transplantation, Department of Surgery, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA ANGELA KERTON • Central Biomedical Services, Imperial College, London, UK HISATAKA KOBAYASHI • Molecular Imaging Program, National Cancer Institute, National Institute of Health, Center for Cancer Research, Bethesda, MD, USA B. CHRISTOFFER LAGERHOLM • MEMPHYS, University of Southern Denmark, Odense, Denmark EDUARDO R. LAZAROWSKI • Cystic Fibrosis/Pulmonary Research and Treatment Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA GARY D. LUKER • Departments of Radiology and Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI, USA KATHRYN E. LUKER • Departments of Radiology and Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI, USA JIAN LUO • Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA, USA RALPH P. MASON • Cancer Imaging Program, UT Southwestern, Dallas, TX, USA LYNN MCKEOWN • Faculty of Life Sciences, The University of Manchester, Manchester, UK LAURA MEZZANOTTE • Department of Pharmaceutical Sciences, University of Bologna, Bologna, Italy ELISA MICHELINI • Department of Pharmaceutical Sciences, University of Bologna, Bologna, Italy TETSUO NAGANO • Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan SEIKO F. OKADA • Cystic Fibrosis/Pulmonary Research and Treatment Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA TAKEAKI OZAWA • Department of Chemistry, School of Science, The University of Tokyo; Japan Science and Technology Agency, Tokyo, Japan KEVIN D.G. PFLEGER • Western Australian Institute for Medical Research (WAIMR) and Centre for Medical Research, University of Western Australia, Perth, Australia GARRETT R. RETTIG • Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Iowa, Iowa City, IA, USA KEVIN G. RICE • Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Iowa, Iowa City, IA, USA EDMOND RICHER • Cancer Imaging Program, UT Southwestern, Dallas, TX, USA BRIAN D. ROBERTSON • Department of Microbiology, Imperial College, London, UK ME´LANIE ROBITAILLE • De´partement de Biochimie, Universite´ de Montre´al; Department of Pharmacology and Therapeutics, McGill University, Montre´al, QC, Canada ALDO RODA • Department of Pharmaceutical Sciences, University of Bologna, Bologna, Italy LUCIA SEMINARIO-VIDAL • Cystic Fibrosis/Pulmonary Research and Treatment Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA YOSHIO UMEZAWA • Department of Chemistry, School of Science, The University of Tokyo; Research Institute of Pharmaceutical Sciences, Tokyo, Japan

Contributors

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YASUTERU URANO • Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan CARLOS VILLALOBOS • Instituto de Biologı´a y Gene´tica Molecular (IBGM), Universidad de Valladolid and Consejo Superior de Investigaciones Cientı´ficas (CSIC), Valladolid, Spain JOHN VIROSTKO • Vanderbilt University Institute of Imaging Science, Vanderbilt University, Nashville, TN, USA GEORGES VON DEGENFELD • Cardiovascular Research, Bayer Healthcare AG, Wuppertal, Germany ALAN S. WAGGONER • Molecular Biosensor and Imaging Center, Carnegie Mellon University; Department of Biology, Carnegie Mellon University, Pittsburgh, PA, USA TOM S. WEHRMAN • Discoverx Corp, Fremont, CA, USA SIOUXSIE WILES • Department of Infectious Diseases and Immunity, Imperial College London, London, UK KITCHENER D. WILSON • Department of Bioengineering, Stanford University; Molecular Imaging Program at Stanford (MIPS), Stanford University, Stanford, CA, USA JOSEPH C. WU • Molecular Imaging Program at Stanford (MIPS), Stanford University and Department of Medicine, Division of Cardiology, Stanford University School of Medicine, and Department of Radiology, Stanford University School of Medicine, Stanford, CA, USA TONY WYSS-CORAY • Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford; GRECC, VA Palo Alto Health Care System, Palo Alto, CA, USA

Chapter 1 Luminescent Probes and Visualization of Bioluminescence Elisa Michelini, Luca Cevenini, Laura Mezzanotte, and Aldo Roda Abstract Bioluminescence (BL) has revealed an extremely useful analytical tool enabling ultrasensitive detection in biotechnological applications. Following the discovery of luciferin and luciferases, molecular biology techniques allowed the cloning of several luciferases and photoproteins. Among most used BL reporters, we find firefly and click-beetle luciferases, bacterial luciferase, Renilla, Gaussia, and Cypridina luciferases, and calcium-activated photoproteins. According to the specific bioluminescent protein, different substrates and protocols must be applied in the experimental procedure for BL measurement. By conjugating (either chemically or by molecular biology techniques) bioluminescent probes to specific targets, it is in fact possible to track a wide range of events and analytes. To aid investigators in the choice and applications of reporter genes, the materials and methods required for BL measurements and experimental protocols are described. Key words: Bioluminescent proteins, Photinus pyralis luciferase, bacterial luciferase, Gaussia luciferase aequorin, reporter gene.

1. Introduction The typical bioanalytical applications of bioluminescent (BL) proteins include the investigation of protein–protein interactions, protein conformational changes, protein phosphorylation, second-messengers expression and, in general, the study of gene expression and gene regulation (1, 2). The expression of a BL protein can be put under the control of tissue-specific regulatory elements allowing non-invasive imaging of physiological and pathological processes like differentiation, apoptosis, tumor progression, and inflammation, even in a 3D fashion by means of BL tomography, which allows 3D BL source reconstruction (3).

P.B. Rich, C. Douillet (eds.), Bioluminescence, Methods in Molecular Biology 574, DOI 10.1007/978-1-60327-321-3_1, ª Humana Press, a part of Springer Science+Business Media, LLC 2009

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Since BL proteins can be detected down to very low levels, they allow ultrasensitive detection of the target analytes and monitoring of the physiological phenomena under investigation. These BL features, associated to instrumental and technical advancements in miniaturization, enable the analysis of small-volume samples, which leads to the development of miniaturized and high-throughput assays. The principal advantages of BL reporter gene-based assays are their high sensitivity, reliability, convenience, dynamic range, and adaptability to high-throughput screening. The choice of reporter gene, however, depends on the cell line used, the nature of the experiment, and the adaptability of the assay to the appropriate detection method (e.g., single-cell imaging versus well- or plate-based detection). A broad variety of BL proteins with different properties is today available for the most demanding applications. Genetically engineered cells (bacteria, yeasts, or mammalian cells) able to produce a BL signal in response to a target analyte represent powerful analytical tools for environmental, medical, and food analysis, and are characterized by low cost and high rapidity and sensitivity. The cells are modified by introducing a reporter gene fused to a regulatory DNA sequence that is activated only in the presence of the analyte of interest, which thus regulates the reporter gene expression. According to intracellular or secreted expression of the BL protein, different protocols have to be used (Fig. 1.1).

Fig. 1.1. Schematic view of heterologous expression of one or more bioluminescent proteins in a cell system. A cell line is transfected with one or more reporter plasmids expressing a BL protein (e.g., firefly or Gaussia luciferases) under the regulation of a specific promoter; cells are lysed and BL emission is measured in cell extracts after substrate addition to assay intracellular activity (e.g., for firefly or click-beetle luciferases). If the BL protein is secreted into the culture medium (e.g., Gaussia and Cypridina luciferases), BL emission is assayed directly in aliquots of culture medium simply by addition of the substrate.

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2. Firefly Luciferase Among BL reporter proteins, luciferase from the North American firefly Photinus pyralis is by far the most employed. Luciferase does not require any post-translational modification for enzyme activity, and it is not toxic even at high concentrations, being thus suitable for in vivo applications in prokaryotic and eukaryotic cells. Several commercially available luciferase assay formulations have been developed, permitting single-step reporter activity measurements, also including cell lysis. Luciferase BL measurements and experimental setup is described in detail depending on the cellular system used (mammalian, bacterial, or yeast cells). 2.1. Mammalian Expression 2.1.1. Materials

1. Mammalian cell line, e.g., HepG2 cells in MEM (minimum essential medium with Earle’s salts), supplemented with 10% (v/v) fetal calf serum, 2 mM L-glutamine, 0.1 mM non-essential amino acids, MEM vitamins, and antibiotic/antimycotic solution (all materials for cell culture from Invitrogen) 2. Triton1 X-100: 1% solution 3. Solution of Trypsin-EDTA (0.25% Trypsin; 1 mM EDTA 4Na) l

4. Luciferase reporter plasmids (Several vectors are available from Promega and Clontech) 5. Exgen500 (MBI Fermentas, Vilnius, Lituania) 6. 96-well flat-bottom microtiter plate 7. Bright-GloTM luciferase assay system (Promega) to be stored in aliquots at –80C 8. Varioskan Flash spectral scanning multimode reader (Thermo Scientific), Veritas microplate luminometer (Turner Biosystems), Centro LB 960 luminometer (Berthold technologies), or Victor multilabel counter (Perkin-Elmer Wallac, Turku, Finland). 2.1.2. Methods

According to the vector used for luciferase expression different protocols may be used, and a careful optimization of experimental procedure is always required depending on the level of luciferase expression, cell type, and instrumentation features. 1. Cell transfection. Cells are passaged when approaching confluence with trypsin/EDTA and plated in 24-well plates approximately at a density of 2–6  104 the day before transfection. About 50–70% confluent cells in 24-well plates are transfected using 1 mg of DNA in physiological solution and 3.3 mL (6 equivalents) of ExGen 500 per well of 24-well plate according to the manufacturer’s instructions (see Note 1). Although the presence of serum does not affect ExGen500-mediated

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transfection efficiency, a phosphate-buffered saline (PBS) wash is preferable before transfection. Transfected cells are incubated at 37C for 48–72 h. For stable transfection, the appropriate selective agent has to be added and transgene expression monitored 10–15 days after transfection. 2. Preparation of cell extracts. Approximately 48–72 h after transfection, cells are washed twice in PBS and lysed with 200 mL of 1% Triton1 X-100 for 5 min at 25C. Cells are scraped from the culture dish, transferred to a microcentrifuge tube, and centrifuged for 2 min at 12,800g to pellet the cell debris. After centrifugation, 100 mL of supernatant (cell extract) is transferred to a white 96-well plate (or luminometer tubes). 3. Measurement for luciferase activity. Each cell lysate is analyzed by the addition of 100 mL (or an equal volume of cell extract) room temperature Bright-GloTM luciferase assay system (see Note 2). Light acquisition from 5 to 30 s, depending on the luminometer used and the sensitivity required (see Note 3). A number of controls are always necessary for quantification of luciferase activity. Positive and negative controls must always be introduced in the experimental setup. The light units values must be corrected for cell number and/or total amount of protein. 2.2. Firefly Luciferase Assay in Bacterial and Yeast Cells

1. Saccharomyces cerevisiae cells expressing firefly luciferase (without the peroxisomal targeting codons, otherwise a cell lysis step is required for the assay) or alternatively bacterial cells expressing firefly luciferase.

2.2.1. Materials

2.

D-luciferin

(Synchem). Working solution: 1 mM in 0.1 M Na citrate buffer (pH 5.0) prepared by mixing 35.0 mL of 0.1 M citric acid and 65.0 mL of 0.1 M trisodium citrate.

3. LB broth medium or yeast synthetic complete (SC) medium. 4. Victor multilabel counter (Perkin-Elmer Wallac, Turku, Finland). 2.2.2. Methods

1. Yeast culture. A 5-mL preculture expressing firefly luciferase is grown overnight at 30C in an orbital shaker in synthetic complete medium. The culture is diluted to OD600 nm ¼ 0.6. The diluted culture is grown at 30C until it reaches midlogarithmic phase (OD600 nm ¼ 1.4). Then 100-mL aliquots of cell culture are pipetted into a 96-well plate. 2. Bacterial cultures. A 5-mL preculture expressing firefly luciferase is grown overnight in LB broth medium at 37C. The culture is diluted to OD600 nm ¼ 0.6. Then 100-mL aliquots of cell culture are pipetted into a 96-well plate for luminescence measurements.

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3. Luminescence measurements. D-Luciferin (1 mM, 100 mL) in 0.1 M Na citrate buffer (pH 3.0 or 5.0) is pipetted into the wells containing 100 mL-aliquots of yeast or bacterial cultures. The plate is briefly shaken and then immediately measured using a luminometer (1–5 s integration time). The light-emission levels are expressed as RLU (relative light units ¼ luminescence value given by the luminometer) (see Note 4).

3. Bacterial Luciferases (lux) Bacterial luciferase catalyzes the oxidation of reduced flavin mononucleotide (FMNH2) and a long-chain aldehyde by molecular oxygen to yield FMN, the corresponding acid, H2O, and light (490 nm)(4). In luminous bacteria the formation of bioluminescent system is controlled by an ‘‘autoinducer’’ (e.g., N-acyl homoserine lactone, AHL) that is produced and secreted by the cells; the inducer levels in the culture medium increase with the growth of the bacterial cells until they reach a threshold and the biosynthesis of bioluminescent system begins (5). 3.1. Materials

Light-producing bacteria (Xenogen)

3.2. Methods

Bacterial luciferase operon contains the genes encoding both for luciferase and for the enzymes able to synthesize its bioluminescent substrate, thus eliminating the need for exogenous substrate addition. Luminous bacteria are suitable for direct determination of sample general toxicity, measuring the decrease of bioluminescent emission as the concentration of toxic compound increase. Due to the low expression levels of bacterial luciferase in mammalian cells, it is mostly used in bacterial BL bioassays. Luciferase assay can be performed in a 96-well microtiter plate: 1. Grow cell culture until OD600 nm ¼ 0.6–0.8. 2. Transfer the cell suspension (200 mL) into each well. 3. Add 100 mL of the sample to be tested to each well in duplicate. 4. Incubate for 30–60 min at 37C. 5. Read with luminometer, 10-s integration time (see Note 5).

4. Gaussia Luciferase Gaussia luciferase (Gluc) is a novel secreted luciferase, cloned from the copepod Gaussia princeps, which catalyzes the oxidation of the small molecule coelenterazine (CTZ) to produce light. Unlike the

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firefly luciferase systems, these CTZ-utilizing luciferases do not require accessory high-energy molecules such as ATP, simplifying their use in a number of reporter applications (6, 7). This luciferase catalyzes the oxidation of the substrate CTZ in a reaction that emits light (lmax ¼ 470 nm), and has considerable advantages over other reporter genes. Since Gluc, when expressed into mammalian cells, is secreted into the culture medium, the BL measurements are performed simply by addition of CTZ in culture medium, without the need for cell lysis. 4.1. Materials

1. Native coelenterazine (Nanolight Technology, Prolume Ltd., Pinetop, AZ). CTZ stock solution is prepared at 1 mg/mL concentration in methanol acidified by HCl (to 10 mL of 100% anhydrous methanol, 50 mL concentrated HCl are added). CTZ is dissolved in an amber microcentrifuge tube to protect from light. CTZ stock solution can be stored at 70C (2–4 week shelf life). Dry CTZ may be stored at 70C for longest storage life. CTZ working solution: CTZ stock solution is diluted to a final concentration of 5 mM in PBS (see Note 6). 2. Vectors expressing the humanized version of Gluc for cloning promoter sequences to assess their transcriptional regulatory functions in mammalian cells (e.g., pGluc-Basic-1 vector from New England BioLabs; pCMV-Gluc-1 from Nanolight Technology) or the native form (pUC19 GLuc from Nanolight Technology) for bacterial expression.

4.2. Methods

Luminescence measurements. CTZ working solution is pipetted into the wells containing yeast, bacterial, or mammalian cells expressing Gaussia luciferase. In order to perform repetitive measurements and real-time monitoring of Gluc expression in the same cellular population, aliquots of culture medium (usually up to 10 mL) are transferred into a 96-well plate and assayed for Gluc emission. The plate is briefly shaken and then immediately measured after CTZ addition using a luminometer with an automated injection system.

5. Cypridina Luciferase Cypridina luciferase is a secreted bioluminescent protein (62 kDa) cloned from the ostracod Cypridina noctiluca, which catalyzes the oxidation of its luciferin to produce light, with a maximum wavelength emission at 465 nm (8).

Luminescent Probes

5.1. Materials

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1. Vector containing Cypridina luciferase cDNA sequence (pCL). 2. Native Cypridina luciferin (also known as Vargulin, NanoLight Technology): 0.5 mM in 10 mM Tris-HCl, pH 7.4.

5.2. Methods

This luciferase has been used as bioluminescent reporter enzyme in yeast, bacterial, and mammalian cell-based assays, with the methods and the same advantages of Gaussia luciferase. Luminescence measurements. Cypridina luciferase activity is measured by mixing 20 mL of culture medium with 80 mL of native or synthetic luciferin. BL measurements are performed by automatic injection of Cypridina luciferin (see Note 7).

6. Aequorin Aequorin is a photoprotein, isolated from the luminescent jellyfish Aequorea victoria, composed of two distinct units, the apoprotein apoaequorin (22 kDa) and the prosthetic group CTZ that reconstitute spontaneously in the presence of molecular oxygen, forming the functional protein (9, 10). Aequorin has become a useful tool in molecular biology for the measurement of intracellular Ca2+ levels, since it has several binding sites for Ca2+ ions responsible for protein conformational changes that convert through oxidation its prosthetic group, CTZ, into excited coelenteramide and CO2. As the excited coelenteramide relaxes to the ground state, blue light (lmax ¼ 469 nm) is emitted and can be easily detected with a luminometer (Fig. 1.2). 6.1. Materials

1. Native coelentarazine (NanoLight Technology, Prolume Ltd., Pinetop, AZ) or Coelenterazine h, a derivative of native coelenterazine that is more sensitive to calcium, making it the ideal choice for luminescent calcium HTS. CTZ working solution: 5 mM CTZ in 100 mM Tris, 90 mM NaCl, 5 mM EDTA, pH 8. 2. Vector expressing mature Aequorin apoprotein sequence in pUC19 vector (e.g., pAPHO from NanoLight Technology). 3. Triggering solution: 0.75% Triton X-100, 15 mM CaCl2.

6.2. Methods

CTZ freely permeates cell membranes, facilitating the reconstitution of the aequorin complex in vivo. Luminescence measurements. 100-mL aliquots of cell suspension (yeast, bacterial, or mammalian cell cultures expressing apoaequorin) in exponential growth phase are pipetted into the wells of

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Fig. 1.2. Schematic view of aequorin BL emission. Aequorin is composed of an apoprotein (molecular mass 21 kDa) and a hydrophobic prosthetic group, CTZ (molecular mass 400 Da). Its polypeptide sequence includes three high-affinity Ca2+ binding sites. Ca2+ binding causes the rupture of the covalent link between the apoprotein and the prosthetic group; this reaction is associated with the emission of one photon.

a 96-well microtiter plate. The cells are incubated overnight with 50 mL of CTZ working solution at 4C in the dark to reconstitute aequorin. Then the plate is warmed at room temperature for 10 –min, and BL emission is measured using a luminometer with an automated injection system. To trigger the reaction, 100 mL of Triggering X-100 solution are added to each well and measured with 1-s integration time.

7. Multiplexed Bioluminescence The recent availability of new reporter genes with improved BL properties, together with technical improvements, prompted the development of multiplexed cell-based assays and multicolor in vivo imaging. We reported dual and triple-color mammalian assays, which combine spectral unmixing of green- and red-emitting luciferases with a secreted luciferase requiring a different substrate, thus allowing to measure three separate targets with high sensitivity and rapidity (11).

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1. Elution buffer: 50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 7. 2. Purified BL proteins requiring the same substrate and emitting at different wavelength like green CBG99luc and red CBRluc genes from Promega in elution buffer. 3.

D-Luciferin

(Biosynth, A.G., Switzerland).

4. Adenosine 50 -triphosphate magnesium salt from bacterial source. 5. 25 mM Glycylglycine buffer (pH 7.8). 6. Eclipse spectrofluorometer (Varian). When two proteins that require different substrates for BL emission are used (e.g., firefly and Renilla luciferase from Promega) in the dual assay: 1. Dual-GloTM Luciferase Assay System (Promega) designed to allow high-throughput analysis of mammalian cells containing genes for firefly and Renilla luciferases, grown in 96- or 384-well plates. 2. ModulusTM Microplate Luminometer (Turner Biosystems) with dual injectors or VeritasTM Microplate Luminometer. 7.2. Methods

Ideally, in a dual-color system the emission spectra of the two reporters would not overlap. Unfortunately, two BL proteins requiring the same substrate whose emissions do not overlap at all have not been identified yet. To minimize spectral overlap, the two emitters should have the narrowest bandwidths possible and wellseparated emission maxima (see Note 8). When two luciferases that emit at different wavelength (e.g., green- and red-emitting firefly luciferases) are used in the same cell-based assay and filters are used to resolve the two signals, a preliminary measurement of the filter correction factors has to be performed by assaying each purified luciferase separately with no filters, and with the green and red filters. These values provide the calibrations constants for the Promega Chroma-Luc calculator, an Excel spreadsheet designed to calculate corrected luminescence values from samples containing red- and green-emitting proteins (12). As previously reported, the concept of detection limit in a dual-color assay is not easy to define (13). In fact, the luminescent signal from one emitter (e.g., green) transmitted through the filter used to monitor the other emitter (e.g., red), i.e. the interference, must be taken into consideration together with the background noise when calculating the detection limit. This interference is concentration-dependent, meaning that the detection limits and the working range of an emitter are dependent on the concentration of the other. 1. Measurement of bioluminescence emission spectra. Emission spectra are obtained using an Eclipse spectrofluorometer (Varian) in ‘‘Bio/Chemiluminescence’’ mode (excitation

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source turned off). Reaction mixtures containing purified green- and red-emitting luciferases (5–100 mg) in elution buffer, 70 mM D-luciferin, and 2 mM Mg-ATP are brought to a final volume of 1 mL with 25 mM glycylglycine buffer (pH 7.8) (see Note 9). Approximately 1 min after initiation of bioluminescence, spectra are recorded in a 1.0-mL fluorescence cuvette and emission slit of 10 nm. Bandwidths of emission spectra are measured at 50% and 20% of the intensity at the maximum wavelength. 2. Bacterial expression and model reporter systems. Dual-reporter model systems were developed to investigate the best luciferase pair for dual reporter assays. The BL proteins are first expressed in 5 mL LB medium cultures of Escherichia coli strain JM109 grown at 37C overnight and diluted in 20 mL LB medium to midlog phase (A600 nm ¼ 0.6). Different proportions of cell cultures expressing the luciferases are transferred in a total volume of 100 mL in a 96-well microtiter plate. All combinations have to be tested in triplicate. 3. Spectral resolution. Luminescence measurements are performed with a Luminoskan Ascent equipped with an injector for substrate addition. An amount of 100 mL of D-luciferin 1 mM in 0.1 M Na citrate buffer solution at pH 5.0 is injected with an automatic dispenser, and after a brief shaking luminescence measurements are performed with 5-s integration. Luciferase activities are measured in the absence or presence of two emission filters. The Promega Chroma-Luc ‘‘Calculator’’ is used to determine the contributions of red- and greenemitting luciferases. Figure 1.3 shows BL emissions obtained

Fig. 1.3. BL emissions of a red-emitting luciferase (&) and a green-emitting luciferase (~) expressed in Escherichia coli JM109 cells grown at 37C. The assay is performed in 96-well microtiter plates using different proportions of two bacterial populations expressing the red- or the green-emitting luciferase. The total cell culture volume is held constant (100 mL). Mean values are plotted, with standard deviations indicated by error bars. RLU, relative light units.

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mixing populations of E. coli JM109 cells expressing a red- and a green-emitting luciferase grown at 37C. Different proportions of cell cultures expressing the luciferases are transferred in a total volume of 100 mL in a 96-well microtiter plate. All combinations are tested in triplicate. Simultaneous measurements of green- and red-emitters are performed in intact E. coli cells in a high-throughput 96-well microplate format. The same assay can be performed using purified BL proteins instead of bacterial cells expressing the two proteins (13).

8. Notes 1. High-quality DNA is critical for successful transfection; an OD260/OD280 ratio of 1.8 or greater is recommended; DNA should be sterile and free of any contaminant such as endotoxins. 2. Since luciferase activity is temperature-dependent, the temperature of the luciferase assay buffers should be held constant at room temperature while quantifying luminescence. Reagent stored frozen after reconstitution must be thawed below 25C to ensure reagent performance. Mix well after thawing. The simplest method for thawing is placing the reagent in a water bath at room temperature. For maximum reproducibility, cell cultures should be equilibrated to room temperature before adding reagent. 3. Using a Luminoskan Ascent luminometer (ThermoLabsystem), a 10-s signal acquisition should be enough when measuring P. pyralis luciferase BL emission in mammalian cells. When higher sensitivies are required (e.g., in case of low levels of luciferase expression), the Bright-GloTM Reagent provides at least fourfold more light output than other extended half-life luciferase reagents. The SteadyGlo1 Luciferase Assay System (Promega) is designed to provide long-lived luminescence (over 5 h) when added to cultured cells and is thus suitable for high-throughput analysis with good reproducibility. 4. When analyzing analytes or liquid samples, it is preferable to add 5 mL of the analyte in solution to 95 mL-cell culture aliquots and add 100 mL of D-luciferin for the light measurement. In the latter case, a blank well should be prepared containing 5 mL of the analyte solvent and 95-mL cell culture aliquots to assess aspecific matrix or solvent effect on the BL signal.

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5. Note that intracellular redox state and growth phase play an important role in the overall response of whole-cell biosensors based on the bacterial luciferase operon; thus studies of the kinetics of induction, as opposed to end-point measurements, are needed to obtain more reliable determinations, especially when high-throughput screening applications are envisioned (14). 6. CTZ spontaneously decays and is unstable for prolonged periods in aqueous solutions. For best results and highest sensitivity, CTZ working solution should be prepared fresh. It is recommended to let the CTZ working solution sit for 15–20 min at room temperature before use. 7. Note that Cypridina luciferin is hundred times more unstable than native coelenterazine, so for best results dissolve immediately before use. 8. Since firefly luciferases are pH-sensitive and may change emission wavelength at different pH, the pH should be measured to investigate if an eventual red-shifting or spectrum broadening caused by pH lowering could interfere with the signal separation. 9. Protein purification may be easily performed by expressing the BL proteins as his-fusion proteins. Briefly, 6his-fusion proteins are first grown in E. coli strain BL-21 in 5-mL LB medium with 100 mg/ml ampicillin at 37C overnight. These cultures are used to inoculate 250 ml cultures at a 1:100 dilution (LB broth supplemented with 100 mg/ml ampicillin), and grown at 37C with shaking until an OD600 nm of 0.6 is reached. Cultures are transferred to a 22C incubator, allowed to equilibrate, and induced with 0.1 mM isopropylbeta-D-thiogalactopyranoside (Sigma) overnight. Qiagen Ni-NTA resins (Qiagen) are used for protein purification according to manufacturer’s instructions, with slight modifications. Cells are harvested by centrifugation, resuspended in 2 ml of lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, 1 mM PMSF, pH 7), and sonicated using ten 10-s bursts with a 15-s cooling period on ice between each burst. The lysate is then centrifuged at 2,200g for 1 h at 4C to pellet cellular debris, and the supernatant is saved to proceed with protocol for purification under native condition. The cleared lysate is mixed with 1 mL of the 50% Ni-NTA slurry, loaded into a polypropylene columns (Qiagen), and washed twice with 4 mL wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, 1 mM PMSF, pH 7). Then 500-mL aliquots are eluted in Elution Buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 7). Protein concentration is determined by Bio-Rad Microassay procedure using bovine serum albumin (BSA) as

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standard. The activity of the purified proteins is evaluated with a luminometer (Luminoskan Ascent, Labsystem) using 4 mL of eluted protein, 100 mL PBS, and 100 mL of BrightGloTM Luciferase Assay System (Promega). References 1. Roda, A., Pasini, P., Mirasoli, M., Michelini, E., and Guardagli, M. (2004) Biotechnological applications of bioluminescence and chemiluminescence. Trends Biotechnol 22, 295–303. 2. Roda, A., Guardagli, M., Pasini, P., and Mirasoli, M. (2003) Bioluminescence and chemiluminescence in drug screening. Anal Bioanal Chem 377, 826–833. 3. Sato, A., Klaunberg, B., and Tolwani, R. (2004) In vivo bioluminescence imaging. Comp Med 54, 631–634. 4. Nealson, K. H., and Hastings, J. W. (1979) Bacterial bioluminescence: its control and ecological significance. Microbiol Rev 43, 496–518. 5. Dunlap, P. V. (1999) Quorum regulation of luminescence in Vibrio fischeri. J Mol Microbiol Biotechnol 1, 5–12. 6. Tannous, B. A., Kim, D. E., Fernandez, J. L., Weissleder, R., and Breakefield, X. O. (2005) Codon-optimized Gaussia luciferase cDNA for mammalian gene expression in culture and in vivo. Mol Ther 11, 435–443. 7. Verhaegent, M., and Christopoulos, T. K. (2002) Recombinant Gaussia luciferase. Overexpression, purification, and analytical application of a bioluminescent reporter for DNA hybridization. Anal Chem 74, 4378–4385. 8. Nakajima, Y., Kobayashi, K., Yamagishi, K., Enomoto, T., and Ohmiya, Y. (2004)

9.

10.

11.

12.

13.

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cDNA cloning and characterization of a secreted luciferase from the luminous Japanese ostracod, Cypridina noctiluca. Biosci Biotechnol Biochem 68, 565–570. Shimomura, O. (2005) The discovery of aequorin and green fluorescent protein. J Microsc 217, 1–15. Shimomura, O. (1995) Luminescence of aequorin is triggered by the binding of two calcium ions. Biochem Biophys Res Commun 211, 359–363. Michelini, E., Cevenini, L., Mezzanotte, L., Ablamsky, D., Southworth, T., Branchini, B., and Roda, A. (2008) Spectral-resolved gene technology for multiplexed bioluminescence and highcontent screening. Anal Chem 80, 260–267. Promega Corporation. (2003) Chroma-Glo Luciferase Assay System Technical Manual No. TM062, 7–9. Branchini, B. R., Southworth, T. L., Khattak, N. F., Michelini, E., and Roda, A. (2005) Red- and green-emitting firefly luciferase mutants for bioluminescent reporter applications. Anal Biochem 345, 140–148. Galluzzi, L., and Karp, M. (2007) Intracellular redox equilibrium and growth phase affect the performance of luciferase-based biosensors. J Biotechnol 127, 188–198.

Chapter 2 Validation of Bioluminescent Imaging Techniques John Virostko and E. Duco Jansen Abstract Bioluminescence imaging (BLI) is frequently cited for its ease of quantification. This fundamental strength of BLI has led to applications in cancer research, cell transplantation, and monitoring of infectious disease in which bioluminescence intensity is correlated with other metrics. However, bioluminescence measurements can be influenced by a number of factors, among them source location, tissue optical properties, and substrate availability and pharmacokinetics. Accounting for these many factors is crucial for accurate BLI quantification. A number of methods can be employed to ensure correct interpretation of BLI results and validate BLI techniques. This chapter summarizes the use of calibrated light-emitting standards, bioluminescence tomography, and post-mortem validation of luciferase expression for validating quantitative BLI measurements. Key words: Bioluminescence imaging, BLI, luciferase, optical imaging, quantification, validation, optical attenuation, light propagation, bioluminescence tomography.

1. Introduction Bioluminescence imaging (BLI) has been applied to a variety of small animal models to provide a quantifiable assessment of bioluminescent cells. Bioluminescence has been used extensively in cancer research; BLI intensity has been used to assess tumor growth (1) and regression in response to treatment (2, 3). BLI quantification has been correlated with caliper measurements (4), excised tumor weight (5), MRI volume (6), and PET measurements of tumor burden (7). Bioluminescence has also been used to visualize transplanted cells (8). BLI has been employed to track engraftment and survival of a variety of cell types, including bone marrow cells, pancreatic islets, and cardiac allografts (9). BLI measures of transplanted cell survival have been correlated with P.B. Rich, C. Douillet (eds.), Bioluminescence, Methods in Molecular Biology 574, DOI 10.1007/978-1-60327-321-3_2, ª Humana Press, a part of Springer Science+Business Media, LLC 2009

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other metrics of cellular engraftment. For example, BLI measurements of cardiac allograft survival correlates with graft beating scores (10). Similarly, BLI measurements after transplantation of pancreatic islets correlate with the number of islets transplanted (11–13). Bioluminescence has been employed to assess the extent of bacterial and viral infection (14–16). In these studies, bioluminescence measurements have been correlated with viral load (17). Imaging gene expression is another quantifiable application of BLI (18). For example, BLI has been used to quantify VEGF gene expression and correlated with ex vivo measures of protein expression (19).

2. Factors Affecting BLI Measurements 2.1. Optical Attenuation Influences BLI Measurements

While bioluminescence measurements have been correlated with other metrics in numerous studies, BLI quantification can be affected by several factors other than the quantity of bioluminescent cells. Biological tissue attenuates light propagation, influencing the detected bioluminescence intensity. Upon entering a biological medium, photons of light can be absorbed by the tissue components and converted to heat, catalyze a chemical reaction, or be released as fluorescence emission. Molecules that absorb light are known as chromophores; for visible light, hemoglobin and melanin are the principle chromophores. Photons are also scattered in media with spatial fluctuations in density and refractive index, resulting in changes in photon path direction. In biological tissue, discrete particles, such as cell membranes, nuclei, collagen, or other cellular microstructures, can cause photon scattering. The attenuation of light by tissue between a bioluminescent source and the detector will decrease the BLI signal. A practical implication of this light attenuation is that bioluminescence measurements are influenced by source depth: sources at increasing depth pass through more biological tissue, suffer greater optical attenuation, and thus exhibit decreased BLI signal (20–22). For instance, a dim superficial source may appear as strong as a bright but deep source, leading to erroneous interpretation that both sources have equal levels of luciferase. Optical attenuation also determines the minimum detectable number of cells, as the minimum signal necessary for detection increases with source depth (20). The optical attenuation of a biological tissue is determined by the propensity of the tissue to absorb and scatter photons of light, known as the optical properties of the tissue. The optical properties of different biological tissues can vary by several orders of magnitude, depending on the molecular components and structure of

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the tissue (23). The attenuation of a bioluminescent signal depends not only on the source depth but also on the tissue or organ through which the light passes. Thus the anatomical location of the source can influence measurements of BLI intensity: bioluminescent sources underneath highly attenuating organs appear less intense. Bioluminescence attenuation is further complicated by the fact that optical properties of biological tissue are not static; dynamic processes can influence optical attenuation. Hemoglobin has been shown to reduce BLI signal due to the strong absorption of light by hemoglobin (24). Increased blood flow, such as present during tumor angiogenesis or hemorrhaging, can influence BLI quantification (25). Surgical artifacts can also affect optical attenuation. For example, after transplantation of bioluminescent cells, inflammation and scarring in tissue overlying the source can lower the detected bioluminescent signal (22). 2.2. BLI Pharmacokinetics Affect BLI Quantification

The chemoluminescent reaction that produces bioluminescence can be influenced by several mechanisms. The luciferase enzyme found in the firefly, Photinus pyralis, catalyzes bioluminescence through the following reaction (26). Luciferase þ Luciferin þ ATP þ O2Mg2þ ! LuciferaseLuciferin þ AMP þ PPi LuciferaseLuciferin þ AMP þ O2 !Oxyluciferin þ CO2 þ AMP Oxyluciferin ! Oxyluciferin þ hv In the presence of oxygen, magnesium, and ATP, the reaction of the luciferase enzyme with the substrate luciferin yields an electronically excited oxyluciferin. The return of oxyluciferin to its ground state is accompanied by the release of a single photon (27). Thus, in the presence of excess oxygen, ATP, and luciferin, the number of photons emitted is proportional to the number of molecules of luciferase present (28). However, the bioavailability of oxygen, ATP, and luciferin can be limiting factors in the bioluminescence reaction, especially in diseased states. In a study of hepatic tumors BLI was found to correlate with tumor burden in small tumors, but for progressively larger tumor sizes this correlation weakened due to tumor hypoxia (25). Similarly, another study found that BLI measurements tend to underestimate tumor volume for fibrotic and necrotic tumors (29). ATP can be a ratelimiting factor in BLI, a fact that has long been used to assay ATP content in cells (30) and map ATP distribution in tumors (31). Bioavailability of the luciferin substrate can also affect the luciferase reaction. The amount of luciferin substrate reaching bioluminescent sources following systemic administration (4) and cellular

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uptake of luciferin (32) have both been implicated in affecting bioluminescence measurements. The pharmacokinetics of luciferin delivery influences bioluminescence measurements. The luciferase reaction is dynamic, with peak intensity reached 5–20 min after administration of luciferin followed by a gradual decline over an hour (1, 6). The decline in bioluminescence intensity results from the clearance of luciferin, while the firefly luciferase enzyme has a half-life of 3 h (33, 34). There is evidence that the time to peak bioluminescence intensity can be affected by source location, presumably due to differences in substrate delivery (35).

3. Accounting for Optical Attenuation Several techniques can be employed to validate accurate quantification of bioluminescence measurements. As previously discussed, optical attenuation can affect the detected bioluminescent source intensity. In order to accurately quantify BLI measurements, the extent of optical attenuation must be determined. Two methods for determining this attenuation and accounting for it in BLI quantification are outlined below. 3.1. Constant LightEmitting Standards

If the location of a bioluminescent source is known, constant lightemitting standards can be implanted at that site to provide a surrogate marker for bioluminescence (22). Luminescent beads (Mb-Microtec, Bern, Switzerland) consist of glass capillary tubes filled with tritium, which excites a phosphor and isotropically emits constant intensity light. The spectral emission from these sources closely mimics the spectral emission of firefly luciferase. When these beads are placed at the site of a bioluminescent source, they can be used to model light attenuation from that site. As light emission from these beads is affected only by tissue optics and not by any biological factors, the proportion of emitted light that is detected can be calculated to quantify optical attenuation. Luminescence imaging of these beads can be performed dynamically after implantation to capture the effect of surgery on luminescence measurements, as observed after transplantation of bioluminescent cells (22).

3.2. Bioluminescence Tomography

If the precise location of bioluminescent sources is not known, mathematical models of light propagation can be employed to reconstruct bioluminescent source location and intensity. Algorithms that tomographically reconstruct bioluminescent sources are under development, which account for optical attenuation when quantifying bioluminescent source intensity (36–47). A

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number of these techniques take advantage of the fact that light attenuation is a function of wavelength, with optical attenuation by biological tissue inversely related to wavelength. These approaches perform BLI at multiple wavelengths and incorporate this multispectral information to determine source depth (36, 37, 41, 44, 46). A simplified example of this multispectral approach is that a light source with equal levels of short- and long-wavelength light would emit greater levels of long-wavelength light relative to short wavelength at a deeper location. Several models of photon propagation have been employed to model bioluminescence, including the diffusion approximation (37, 39, 41, 44) and Monte Carlo modeling (43). A common attribute of these algorithms is that the optical properties of the biological tissue must be included to model the absorption and scatter of photons from a bioluminescent source. One approach is to model the mouse as optically homogeneous. However, as previously noted, the optical properties of different tissues varies, leading to erroneous reconstruction of bioluminescent sources in heterogeneous mouse tissue when optical homogeneity is assumed (36, 48). Several approaches to model optical heterogeneity are under development. One approach is to use an atlas of mouse organs and assign optical properties based on that atlas (36, 46). However, deformation of the actual mouse volume from that of the atlas may impair accuracy using this method. Another approach is to determine the anatomy from another modality, such as MRI (49). However, this method sacrifices both the speed and inexpensiveness of BLI, and has thus far only been shown to marginally improve reconstruction accuracy (49). The choice of optical properties to assign to each organ also influences reconstruction accuracy, as published values can vary significantly (23). In situ measurements of optical properties have been shown to improve BLI reconstruction accuracy (38).

4. Substrate Availability and Pharmacokinetics

As previously discussed, substrate availability and pharmacokinetics can also influence bioluminescence measurements. Consistent substrate availability requires precise injection of luciferin, with total dosage normalized by the animal weight. The pharmacokinetics of luciferin are dynamic and must be controlled. One study found that the tumor bioluminescence measured 10–20 min after intraperitoneal luciferin administration correlated well with peak luminescence, the intensity integrated for an hour postluciferin administration, and tumor size (4). This imaging window

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10–20 min after luciferin injection seems to provide the most consistent bioavailability of luciferin, but should be validated in each animal model with repeatability studies.

5. Post-mortem Validation of Luciferase Expression

Even after controlling for tissue optical attenuation and substrate availability and pharmacokinetics, BLI measurements can be affected by other factors, such as co-factor availability. Availability of oxygen and ATP can be difficult to non-invasively determine in vivo. However, assays of luciferase content (50, 51) and luciferase immunohistochemistry (52) can be performed post-mortem to ensure that co-factor availability is not influencing bioluminescence measurements.

5.1. Luciferase Staining

Immunohistochemical staining can be used to validate luciferase expression. Rabbit anti-luciferase antibodies, such as that from Cortex Biochem, Inc. (San Leandro, CA), can be employed to detect luciferase on tissue slides. To systematically examine luciferase expression in the tissue sections, sections throughout the tissue block can be stained for luciferase expression. This immunohistochemical validation can then be correlated with in vivo BLI measurements.

5.2. Luciferase Activity

Post-mortem measures of luciferase activity in tissue lysates can be determined by luminometer. These luminometer assays provide luciferin, oxygen, and ATP in excess to prevent any of these components from being rate-limiting in the luciferase reaction. Furthermore, as tissues are homogenized prior to the assay, optical attenuation is negligible. Several studies have correlated in vivo BLI measurements with luminometer measurements of luciferase activity in vitro (50, 51). In applications where protein content can vary from sample to sample, luciferase activity can be normalized to total protein.

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therapies using in vivo bioluminescence imaging. Blood 101, 640–648. 3. Sweeney, T. J., Mailander, V., Tucker, A. A., Olomu, A. B., Zhang, W., Cao, Y., Negrin, R. S., and Contag, C. H. (1999) Visualizing the kinetics of tumor-cell clearance in living animals. Proc Natl Acad Sci USA 96, 12044–12049. 4. Paroo, Z., Bollinger, R. A., Braasch, D. A., Richer, E., Corey, D. R., Antich, P. P., and

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Mason, R. P. (2004) Validating bioluminescence imaging as a high-throughput, quantitative modality for assessing tumor burden. Mol Imaging 3, 117–124. Vooijs, M., Jonkers, J., Lyons, S., and Berns, A. (2002) Noninvasive imaging of spontaneous retinoblastoma pathway-dependent tumors in mice. Cancer Res 62, 1862–1867. Rehemtulla, A., Stegman, L. D., Cardozo, S. J., Gupta, S., Hall, D. E., Contag, C. H., and Ross, B. D. (2000) Rapid and quantitative assessment of cancer treatment response using in vivo bioluminescence imaging. Neoplasia (New York, N.Y.) 2, 491–495. Ray, P., Wu, A. M., and Gambhir, S. S. (2003) Optical bioluminescence and positron emission tomography imaging of a novel fusion reporter gene in tumor xenografts of living mice. Cancer Res 63, 1160–1165. Cao, Y. A., Wagers, A. J., Beilhack, A., Dusich, J., Bachmann, M. H., Negrin, R. S., Weissman, I. L., and Contag, C. H. (2004) Shifting foci of hematopoiesis during reconstitution from single stem cells. Proc Natl Acad Sci USA. 101, 221–226. Cao, Y. A., Bachmann, M. H., Beilhack, A., Yang, Y., Tanaka, M., Swijnenburg, R. J., Reeves, R., Taylor-Edwards, C., Schulz, S., Doyle, T. C., Fathman, C. G., Robbins, R. C., Herzenberg, L. A., Negrin, R. S., and Contag, C. H. (2005) Molecular imaging using labeled donor tissues reveals patterns of engraftment, rejection, and survival in transplantation. Transplantation 80, 134–139. Tanaka, M., Swijnenburg, R. J., Gunawan, F., Cao, Y. A., Yang, Y., Caffarelli, A. D., de Bruin, J. L., Contag, C. H., and Robbins, R. C. (2005) In vivo visualization of cardiac allograft rejection and trafficking passenger leukocytes using bioluminescence imaging. Circulation 112, I105–I110. Chen, X., Zhang, X., Larson, C. S., Baker, M. S., and Kaufman, D. B. (2006) In vivo bioluminescence imaging of transplanted islets and early detection of graft rejection. Transplantation 81, 1421–1427. Fowler, M., Virostko, J., Chen, Z., Poffenberger, G., Radhika, A., Brissova, M., Shiota, M., Nicholson, W. E., Shi, Y., Hirshberg, B., Harlan, D. M., Jansen, E. D., and Powers, A. C. (2005) Assessment of pancreatic islet mass after islet transplantation using in vivo bioluminescence imaging. Transplantation 79, 768–776. Lu, Y., Dang, H., Middleton, B., Zhang, Z., Washburn, L., Campbell-Thompson, M.,

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Chapter 3 Assessment of Extracellular ATP Concentrations Lucia Seminario-Vidal, Eduardo R. Lazarowski, and Seiko F. Okada Abstract Most cells release ATP to the extracellular milieu. Extracellular ATP plays important signaling roles by activating a score of broadly distributed cell surface purinergic receptors (purinoceptors). Biological responses regulated by purinergic receptors include neurotransmission, smooth muscle relaxation and contraction, epithelial cell ion transport, inflammation, platelet activation, immune responses, cardiac function, endocrine and exocrine secretion, glucose transport, and cell proliferation. ATP concentrations at the cell surface, and consequently the magnitude of purinergic receptor stimulation, reflect a wellcontrolled balance between rates of ATP release and extracellular metabolism. Given the broad spectrum of responses triggered by extracellular ATP, there is a growing interest in accurately assessing the concentrations of this nucleotide at the cell surface. In this chapter, we discuss the use of the luciferin/ luciferase-based reaction to measure extracellular ATP concentrations with high sensitivity. Protocols are adapted to assess ATP levels either in sampled extracellular fluids or in situ at the cell surface. Although our focus is on studies of ATP release from epithelial cells, protocols described here are applicable to practically all cell types. Key words: ATP release, extracellular ATP, ecto-ATPase, luciferase, protein A-luciferase, luciferin. Abbreviations: 6  His: hexa-histidine, ALU: arbitrary light unit, ARL-67156: 6-N-N-diethylb,g-dibromomethylene-D-ATP, b,g-metATP: b,g-methyleneadenosine 50 -triphosphate, BSA: bovine serum albumin, DMEM: Dulbecco’s modified eagle’s medium, ebselen: 2-phenyl-1,2benzisoselenazol-3(2H)-one, FBS: fetal bovine serum, HBSS: Hank’s balanced salt solution, HEPES: 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid, MEM: minimum essential medium, PBS: phosphate-buffered saline, RT: room temperature, SPA-luc: Staphylococcus protein A-fused luciferase.

1. Introduction ATP is an essential component of living cells. ATP is the major source of energy in most biosynthetic processes, participates as cofactor or activator of numerous enzymatic reactions, and is a P.B. Rich, C. Douillet (eds.), Bioluminescence, Methods in Molecular Biology 574, DOI 10.1007/978-1-60327-321-3_3, ª Humana Press, a part of Springer Science+Business Media, LLC 2009

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building block of nucleic acid chains. In addition, ATP is released from cells in a regulated manner to accomplish autocrine and paracrine functions via the activation of cell surface purinergic receptors (purinoceptors) (1). Purinoceptors consist of three widely distributed families: P2X, P2Y, and P1 receptors. Purinoceptor-mediated responses include cell proliferation, migration, differentiation, embryonic development, wound healing, restenosis, atherosclerosis, ischemia, turnover of epithelial cells in skin and visceral organs, inflammation, neuroprotection, and cancer (1). P2X receptors are ligand-gated cation channels. They include seven molecularly defined species (P2X1–P2X7), all of which are selectively activated by ATP, but not by other nucleotides (2). The P2Y receptor family includes eight G protein-coupled receptors: the P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14 receptors. The P2Y2 receptor is potently activated by ATP (and UTP). The P2Y11 receptor is selectively activated by ATP, whereas the P2Y12 and P2Y13 receptors are most potently and selectively activated by ADP. P2Y4, P2Y6, and P2Y14 receptors are activated selectively by uridine nucleotides and UDP-sugars (1). The P1 receptor family (A1, A2a, A2b, and A3 adenosine receptors) is activated by the nucleoside adenosine, the final product of ATP dephosphorylation. Cellular ATP is released both in the absence of external stimuli and in response to physiological stimuli. Levels of extracellular ATP are controlled by a complex array of nucleotide-metabolizing cell surface enzymes, which include ecto-nucleotidases of the ectonucleotide triphosphate diphosphohydrolase (eNTPDase) and ecto-nucleotide pyrophosphatase (eNPP) families, 50 -nucleotidase (50 -NT), non-specific phosphatases, and transphosphorylating enzymes, such as nucleoside diphosphokinase and adenylyl kinase (3). Thus, extracellular ATP concentrations and, consequently, ATP, ADP, and adenosine actions on purinergic receptors, are dynamically regulated via cellular release and extracellular metabolism of ATP (4). Given the physiological importance of purinergic signaling, there is an increased interest in assessing nucleotide concentrations on the surface of cells and tissues and in understanding the mechanisms of cellular ATP release. Numerous approaches have been developed in recent years to assess extracellular levels of ATP and other nucleotides (reviewed in (5)). Several factors complicate the accurate measurement of extracellular ATP concentrations. For example, it is difficult to assess ATP concentrations in the physiologically relevant unstirred film covering the cell surface. Moreover, robust ATP release occurs in response to mechanical stress; thus, experimental maneuvers (cell wash, sampling, transporting the cell dishes) often result in artifacts. Finally, rapid hydrolysis of released ATP may compromise the relevance of ATP measurements.

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In this chapter, we will describe approaches to measure ATP concentrations in sampled, diluted extracellular fluids, as well as in cell surface thin films (Fig. 3.1). We will focus on epithelial cells as examples; however, these methods are applicable to all cell types. A

B

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hv

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luciferin luciferase

Luminometer

Fig. 3.1. Off-line and real-time approaches to measure extracellular ATP concentrations. Extracellular ATP concentrations can be measured by off-line luminometry of sampled extracellular fluids (A), or on-line luminometry using either soluble luciferase dissolved in medium covering the cells (B) or cell –surface-attached luciferase (C). ATP concentrations detected by each method in different volumes are illustrated in Figs. 3.2 and 3.3.

1.1. Measuring ATP Concentrations in Sampled Fluids: OffLine Bioluminescence Detection

This section describes a protocol that uses the luciferin/luciferasebased reaction (see Note 1) to quantify ATP concentrations in samples obtained from cell culture-conditioned media. In epithelial and endothelial cells, robust ATP release can be triggered by mechanical stimuli such as shear stress, stretch, compression, and hypotonicity-induced cell swelling (4, 6–9). Here, we will use hypotonicity-induced ATP release as an example. ATP release can also be measured after inhibition of ATP metabolism. Commonly used inhibitors of ecto-nucleotidase activities are b,g-methyleneadenosine 5-triphosphate (b,g-metATP), 6-N-N-diethyl-b,gdibromomethylene-D-ATP (ARL-67156), and 2-phenyl-1,2benzisoselenazol-3(2H)-one (ebselen) (4, 8–11). Levamizole has been used to inhibit alkaline phosphatase activity present on epithelial cells (4). In this example, we obtained maximal inhibition of ATP metabolism in A549 cell cultures by using a cocktail of b,g-metATP and ebselen. After stimulation of the cells and/or inhibition of ATP metabolism, the conditioned media are analyzed for ATP concentration. Briefly, samples are collected gently to minimize unwanted mechanical release of ATP, boiled to abolish ATPase activities potentially present in the extracellular solution, and transferred to the dark chamber of a luminometer. The luciferase/luciferin cocktail is added by an automatic injector, and the resulting luminescence is recorded (Fig. 3.1A). The methodology described here is applicable to ATP measurements in tissue extracts, biological fluids, bacterial cultures, in vitro enzymatic reactions, etc.

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1.2. Real-Time, Cell Surface Measurement of Extracellular ATP

In this section, we describe methods for real-time measurement of ATP by using cell surface-bound luciferase (Fig. 3.1C), and will compare this method with measurements obtained with soluble luciferase (Fig. 3.1B). The protocols below are designed for measuring luminal ATP concentrations on polarized epithelial cells; however, they are also applicable to measuring extracellular ATP concentrations of non-polarized cells grown on culture plates. Cell surface-binding luciferase can be engineered by fusing luciferase to cell surface-binding constructs, e.g., Staphylococcus protein A (4, 10, 12), biotin, or lectins, and allows the assessment of ATP concentrations near the cell surface. Soluble luciferase assesses the average ATP concentrations in the medium (from the cell surface to the surface of the bathing solution) and, when used in a small volume, reflects near-cell surface ATP concentrations (Figs. 3.2 and 3.3). For real-time assessment of ATP concentrations, cultures (either non-polarized or polarized) are placed directly in the luminometer. Sampling Real-time (Soluble luciferase) Real-time (Cell-attached luciferase)

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Fig. 3.2. Basal ATP concentrations on the cell surface. ATP concentrations in varied luminal volumes on resting human bronchial cells were measured by off-line luminometry (as in Fig. 3.1A, grey triangle), or by real-time measurement with luciferase dissolved in bulk (as in Fig. 3.1B, open circle), and attached to the cell surface (as in Fig. 3.1C, solid diamond). Values are mean – SEM of four Transwells/subject established from three different subjects. No major differences in basal ATP concentrations were observed with these approaches. Reprinted with permission from JBC, vol. 281, no. 32, pp. 22992– 23002 (2006).

2. Materials All reagents should be of the highest purity available and maintained free of bacterial contamination to avoid ATP degradation. Use of aerosol-protected tips is strongly recommended to avoid reagent cross-contamination.

Assessment of Extracellular ATP Concentrations

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Fig. 3.3. Hypotonicity-induced ATP release. Primary human bronchial epithelial cultures were exposed to luminal 33% hypotonic challenge at t = 0. ATP concentrations were measured by off-line luminometry (A), real-time luminometry with soluble luciferase (B), and real-time luminometry with cell surface-attached luciferase (C). Varied luminal volumes were applied on 12-mm Transwells, as indicated. D: Summary data illustrating peak ATP concentrations as measured by soluble luciferase (open circle) and cell-attached luciferase (solid diamond) in varied luminal volumes. In (A)–(C), values are mean – SEM of 3–4 Transwells/subject established from three different subjects. In diluted solutions (100–500 ml), ATP concentrations measured at the cell surface (C) are higher than those measured in bulk (B) or by sampling (A). However, ATP concentrations in small volumes (25–50 ml) were similar between cell-attached luciferase detection and soluble luciferase detection (D). Reprinted with permission from JBC, vol. 281, no. 32, pp. 22992–23002 (2006).

2.1. Measuring ATP Concentrations in Sampled Fluids: Offline Bioluminescence Detection 2.1.1. Cell Culture 2.1.2. Stimulation of ATP Release by Hypotonic Swelling

Experiments illustrated in this section are performed with A549 cells (ATCC # CCL-185) seeded on 24-well multiwell plastic plates (BD Falcon). Cells are grown on Dulbecco’s modified eagle’s medium (DMEM) with high glucose (D-Glucose: 4.5 g/L), supplemented with 10% fetal bovine serum (FBS), 60 mg/ml (100 IU/mL) penicillin, and 100 mg/mL streptomycin. 1. Hypotonic solution: 1.2 mM CaCl2, 1.8 mM MgCl2, and 25 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), pH 7.4. Store at 4C. 2. Control (isotonic) solution: 154 mM NaCl (0.9% NaCl solution), 1.2 mM CaCl2, 1.8 mM MgCl2, and 25 mM HEPES, pH 7.4. Store at 4C.

2.1.3. Inhibition of ATP Metabolism

1. Ebselen: 10 mM in dimethyl sulfoxide (DMSO), aliquoted, and stored at –20C. 2. b,g-metATP: 100 mM in water, aliquoted, and stored at –20C.

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In A549 cell cultures, we obtain maximal inhibition of ATP metabolism by using a cocktail containing 300 mM b,g-metATP and 30 mM ebselen (see Fig. 3.4). A

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Fig. 3.4. Effect of pharmacological reagents on ATP detection. (A) Luciferase activity is not affected by ATPase inhibitors, but decreases in the presence of some purinoceptor antagonists. Calibration curves of ATP were performed in HBSS+ alone, or supplemented with 300 mM b,g-metATP, or 30 mM ebselen. Inset: 100 nM ATP was prepared in HBSS+ alone, or containing 100 mM pyridoxal-phosphate-6-azophenyl-2’,4’-disulfonic acid (PPADS), 100 mM reactive blue 2 (RB2), or 100 mM suramin. Values are the mean – SEM of three separate experiments, n = 3. (B) Effect of ecto-ATPase inhibitors on ATP hydrolysis in A549 cells. Lung alveolar A549 cells were incubated for the indicated times at 37C with 300 ml HBSS+ containing 100 nM ATP (vehicle), or 100 nM ATP and 300 mM b,g-metATP, or 100 nM ATP and 30 mM ebselen, or 100 nM ATP and b,g-metATP and ebselen combined. Samples were collected and luminescence recorded as described in the Methods section. Values are the mean – SEM of two separate experiments, n = 4. (C) Measurements of extracellular ATP concentrations are underestimated in the absence of ecto-ATPase inhibitors. Confluent A549 cells grown on 24-well plates were incubated at 37C for 5 min with 300 ml HBSS+ in the absence (control) or in the presence of 300 mM b,g-metATP and 30 mM ebselen. Cultures were subsequently treated for 5 min with isotonic solution or 33% hypotonic challenge. Samples were collected and luminescence recorded as described in the Methods section. Values are the mean – SEM of 2 separate experiments, n = 6. 2.1.4. Luminometry Reagents

Several commercial brands of luminometers are available. The protocol described below was adapted for a Berthold AutoLumat luminometer, which is configured to process 180 test tubes at a time (see Note 2). 1. 4X LUMI solution: 6.25 mM MgCl2, 0.63 mM ethylenedinitrilotetraacetic acid (EDTA), 75 mM dithiothreitol (DTT), 1 mg/mL bovine serum albumin (BSA), and 25 mM HEPES, pH 7.8. Filter and store sterile at 4C. 2. Luciferase from Photinus pyralis (Sigma) is dissolved at 0.5 mg/ mL in 4X LUMI solution and stored in 30 ml aliquots at –20C. 3. Luciferin (BD PharMingen) is dissolved at 10 mg/mL in water and stored in 100 mL aliquots, protected from light at –20C. 4. Hank’s balanced salt solution (HBSS) supplemented with 1.2 mM CaCl2 and 1.8 mM MgCl2 (HBSS+). HBSS+ is filtered sterile and stored at 4C. 25 mM HEPES, pH 7.4, is added freshly prior to experiments (see Note 3).

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5. 5 mL clear polystyrene or glass test tubes (e.g., Sarstedt). 6. ATP stock solution (e.g., 100 mM, GE Healthcare) stored at –20C. 2.2. Real-Time, Cell Surface Measurement of Extracellular ATP

1. Cells can be grown on plastic dishes (for non-polarized cells) or Transwells (for polarized cells) 3.5 cm or less in diameter. 2. Luciferase (Sigma) 3. Luciferin (BD PharMingen) 4. Staphylococcus protein A-fused luciferase (SPA-luc; modified from the original construct provided by Dr. George Dubyak, Case Western Reserve University; see Note 4 and (4) for purification protocols) 5. Blocking solution: PBS containing 1% BSA (PBS/BSA) 6. Anti-keratan sulfate antibody (mouse IgG2b, Chemicon, Temecula, CA) 7. Buffer: HBSS+ buffered with 10 mM HEPES (HBSS/ HEPES). HBSS+ can be replaced with other nutrient-containing solutions (e.g., DMEM, MEM, F12). 8. Luminometer with a real-time measurement function, e.g., TD-20/20 (Turner Biosystems, Sunnyvale, CA).

3. Methods 3.1. Measuring ATP Concentrations in Sampled Fluids: OffLine Bioluminescence Detection 3.1.1. Preparation of Samples

1. Grow lung epithelial A549 cells in 24-well plastic plates (surface area of 2 cm2) until confluence (see Note 5). 2. Rinse confluent cultures gently twice with HBSS+ to remove cell debris and serum components present in the medium. 3. Pre-incubate cells in HBSS+ for 1 h at 37C and 5% CO2 in a tissue culture incubator. To minimize unwanted mechanically induced ATP release during sampling, cell cultures should be covered sufficiently with media (e.g., 250 mL for each well of a 24-well plate). 4. Expose cell cultures to reagents and/or stimuli, as described in Fig. 3.4. 5. Collect up to 100 mL of the cell bathing medium into 1.5-mL microcentrifuge tubes placed on ice. 6. Heat samples for 2 min at 98C to inactivate potential nucleotidase activities. 7. Store samples at –20C until bioluminescence measurements.

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3.1.2. Quantification of ATP

This protocol assumes the use of a LB953 AutoLumat luminometer (Berthold, Wildbad, Germany), but can be modified to other luminometers by following the manufacturer’s instructions (Figs. 3.4 and 3.5).

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Fig. 3.5. Quantification of ATP using luciferin/luciferase. (A) Anions greatly interfere with the luciferase reaction. Calibration curves of ATP were performed by adding 30 ml of the indicated ATP concentrations to 300 ml H2O or 154 mM NaCl. Values are the mean – SEM of two separate experiments, n = 3. (B) Serum components decrease ATP detection. ATP was diluted at the indicated concentrations in H2O, HBSS+, MEM, or MEM supplemented with 10% FBS. A 30-mL aliquot was collected and added to a 5-mL test tube containing 300 mL of water. Values are the mean – SEM of two separate experiments, n = 4. (C) Albumin and other serum components affect ATP detection. 100 nM ATP was prepared in MEM, MEM supplemented with 10% FBS, or MEM supplemented with 4 g/dL human albumin, and incubated at RT for 10 min. Samples were heated at 98C for 2 min (except non-heated controls) prior to ATP measurements.

1. Prepare the luciferin/luciferase cocktail freshly by adding one aliquot of luciferase and luciferin stock solutions (described in Materials) to 12.5 ml 4X LUMI solutions at room temperature (RT), protected from light. Final luciferin and luciferase concentrations in 4X LUMI are 265 mM and 1.2 mg/ml, respectively. 2. Place the luciferin/luciferase solution in the injector port of the luminometer. Prime the injector line following the manufacturer’s instructions. 3. Prepare an ATP calibration curve (e.g., up to 1,000 nM ATP, see Note 6) in the same solution/media used for incubations with cells. 4. Add 30 mL of each sample to a 5 mL test tube containing 300 mL H2O (see Note 7). 5. Transfer the test tubes to the dark chamber of the luminometer and proceed with the luciferin/luciferase injection and bioluminescence recording, i.e., arbitrary light units (ALUs), as instructed by the manufacturer. 6. Determine ATP concentration in the sample by intersecting sample ALU values with the calibration curve ALU values (see Notes 8 and 9).

Assessment of Extracellular ATP Concentrations

3.2. Real-Time, Cell Surface Measurement of Extracellular ATP 3.2.1. Attachment of Staphylococcus Protein A-Fused Luciferase (SPA-Luc) to Cell Surface (see Note 10)

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1. Wash the surface (apical, if polarized cells are used) of cell cultures with phosphate-buffered saline (PBS), 3  . 2. Incubate the (apical) surface with 50 mL (for cultures of 12 mm diameter) of blocking solution for 30 min on ice. If polarized cells are used, keep the basolateral surface immersed in medium. 3. Replace the blocking solution with a solution containing the designated primary antibody (see Note 11). For primary airway epithelial cells, use 50 mL of 10 mg/mL (i.e., 1:300) antikeratan sulfate antibody in PBS/BSA. Incubate for 1 h on ice. 4. Wash 3  with PBS. 5. Incubate with 0.5 mg/mL purified SPA-luc (see Note 4) for 1 h at 4C in the dark. SPA-luc will bind to the Fc domain of the antibody attached to the cells, as indicated in Step 3. 6. Wash carefully 3  with PBS. Replenish the apical surface with ATP assay solution (e.g., HBSS/HEPES). Keep cultures in the dark at RT for 30 min to equilibrate the extracellular ATP concentrations.

3.2.2. Measurement of Cell Surface ATP Concentrations Using SPA-luc

1. Place a SPA-luc-bound cell culture in the Turner TD-20/20, add soluble luciferin (150 mM final, to the apical solution for polarized cultures) and close the lid. When a Transwell is used, place it in a chamber (or a dish) containing HBSS/ HEPES to cover the basolateral side (Fig. 3.1B, C). Assays are typically performed at RT (see Note 12). 2. Record baseline luminescence (arbitrary light unit, ALU) every minute with 5–10 s integration time, according to manufacturer’s instructions. Monitor ALU until baseline luminescence is achieved (see Note 13). Baseline luminescence is usually achieved within 5–30 min and represents basal ATP concentrations (see Fig. 3.3). 3. To assess stimulated ATP release, add stimuli (e.g., pharmacological reagents, hypotonic challenge, etc.) and record the ALU. ALU integration time needs to be optimized, as well as the frequency of recording, for each experiment. For example, when airway epithelial cells are challenged with 33% hypotonicity, H2O (a half volume of the initial luminal volume) is added to the luminal solution at t = 0. The ALU is recorded for 5 min; every 0.2 s for the first minute, then every 10 s (with 4-s integration time) for the next 4 min. A typical time-course of ATP concentrations is shown in Fig. 3.3. 4. At the end of each assay, an ATP–luminescence relationship (calibration curve) is generated to calculate ATP concentrations. Known concentrations of ATP are added to the luminal liquid in a stepwise manner (e.g., 1 nM added twice, 10 nM added twice, then 100 nM added twice – for the accuracy of

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the calibration curve, adding each concentration twice is recommended), and increases in ALUs recorded each time (see Note 14) 3.2.3. Measurement of Cell Surface ATP Concentrations Using Soluble Luciferase

1. Wash the surface of cultures with PBS,3  . 2. Add HBSS/HEPES (0.5–1 ml for non-polarized 3.5 cm cultures. Bilaterally for polarized cultures – 1 cc and 25–500 ml to luminal and serosal side, respectively, when 12-mm Transwell is used). Equilibrate the cultures in an incubator (37C and 5% CO2) for 1 h. 3. Add luciferase (0.8 mg/cm2 culture surface) and luciferin (150 mM) to the luminal buffer, and start the measurement as described in Methods 3.2.2.

4. Notes 1. Firefly luciferase catalyzes the following reaction: D-luciferin þ ATP þ luciferase (L) ! L(luciferyl-adenylate)

þ pyrophosphate L(luciferyl-adenylate) þ O2 ! L(oxyluciferin*; AMP) + CO2 L(oxyluciferin*; AMP) ! L(oxyluciferin; AMP) þ photon L(oxyluciferin; AMP) ! L þ oxyluciferin þ AMP 2. Many luminometers are configured as microplate readers. Sample volume and luciferase-luciferin cocktail should be modified to fit the volume of an individual well, following the manufacturer’s instructions. 3. Minimum essential medium (MEM), DMEM, or several other culture media (without serum) are equally effective as HBSS+, and could be used as an alternative in the sample preparation assay. Avoid using media supplemented with ATP, such as Medium 199. 4. SPA-luc fused to a hexa-histidine (6  His) tag (4) is purified over a Ni2+-chelating column. The 6  His tag is cleaved by Tobacco-Etch virus (TEV) protease after purification. For detailed purification protocols, see (4). 5. Seeding density of 1  105 A549 cells/well will provide confluent cultures at 24 h. 6. Under the conditions described, a linear ATP concentration:luminescence relationship is observed in the range of 0.1– 1000 nM ATP. This ATP concentration range covers ATP concentrations detected in the bulk extracellular medium of most cell cultures.

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7. Media or other saline-based solutions (e.g., 0.9% NaCl or PBS) contain anions that interfere with the luciferase reaction [Fig. 3.5A and (13, 14)], decreasing the sensitivity of the assay. Therefore, we recommend using water as the diluting agent to achieve the highest sensitivity in the assay. 8. The luciferase reaction is inhibited by components present in cell culture media, e.g., anions [Fig. 3.5B and (14)]. Moreover, phosphatases and other components present in FBS-supplemented and hormone-supplemented media (e.g., BEGM or SAGM, Lonza Walkersville, MD) affect ATP availability for the luciferase reaction. Albumin-bound ATP can be dissociated by heating the sample at 95C for 2 min (Fig. 3.5C). 9. All test drugs added to the cells should be tested for potential interference with luciferase activity [Fig. 3.4 and (14)]. 10. The principle of SPA-luc attachment to cell surface is as follows. First, bind an antibody to cell surface molecules; next, attach protein A (of SPA-luc) to the Fc domain of the antibody. It is important to choose an antibody that protein A is capable of binding; for example, protein A strongly binds to total IgG, IgG2a, IgG2b, and IgG3, but exhibit weak or no binding to IgG1, which is the most common class of monoclonal antibodies. 11. For primary human airway cells, lectins and monoclonal antibodies against keratan sulfate or MUC1 served as SPA-luc attachment molecules (4). For mouse Bac-1.2F5 macrophages, monoclonal antibodies against CD45.2 or H-2 Kd major histocompatibility complex (MHC) class I; for human platelets, monoclonal antibodies against CD41 or anti-HLAABC served as SPA-luc attachment molecules (12). For cell types in which finding an endogenous antigen on the cell surface for sufficient antibody attachment is difficult, antigens can be overexpressed [e.g., CD14 (10)]. However, the effect of antigen overexpression on ATP release and metabolism needs to be addressed. 12. Though it is ideal to perform ATP release assays at a physiological temperature (37C), luciferase activity is dramatically decreased above 30C (15). Being aware that some ATP release pathways (e.g., exocytosis) might be suppressed at low temperatures, assays can be carried out at RT. It is critical to maintain pH of the assay solution on cells (which contains luciferin and luciferase) at 7.0–7.4 (15) by including 25 mM HEPES (pH 7.4). 13. Experimental maneuvers, such as changing and adding luminal solutions and transferring Transwells, cause robust ATP release from cells. Baseline ATP concentrations are achieved after such artifactually released ATP is hydrolyzed by endogenous ecto-ATPases, usually within 5–30 min of incubation.

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14. The sensitivity of luciferin-luciferase reactions may vary among assays; thus, an ATP–ALU relationship should be generated for each assay. The end products of luciferin-luciferase reaction (e.g., pyrophosphate, oxyluciferin) inhibit the luciferase reaction. However, when sufficient amounts of luciferin and luciferase are included at the beginning of the assay, the assay sensitivity is typically maintained for at least 30 min on cells. References 1. Burnstock, G. (2006) Purinergic signalling. Br J Pharmacol 147 (Supple 1), S172–S181. 2. North, R. A. (2002) Molecular physiology of P2X receptors. Physiol Rev 82, 1013–1067. 3. Zimmermann, H. (2000) Extracellular metabolism of ATP and other nucleotides. Naunyn Schmiedebergs Arch Pharmacol 362, 299–309. 4. Okada, S. F., Nicholas, R. A., Kreda, S. M., Lazarowski, E. R., and Boucher, R. C. (2006) Physiological regulation of ATP release at the apical surface of human airway epithelia. J Biol Chem 281, 22992–23002. 5. Lazarowski, E. R., Shea, D. A., Boucher, R. C., and Harden, T. K. (2003) Release of cellular UDP-glucose as a potential extracellular signaling molecule. Mol Pharmacol 63, 1190–1197. 6. Gatof, D., Kilic, G., and Fitz, J. G. (2004) Vesicular exocytosis contributes to volumesensitive ATP release in biliary cells. Am J Physiol Gastrointest Liver Physiol 286, G538–G546. 7. Boudreault, F., and Grygorczyk, R. (2004) Cell swelling-induced ATP release is tightly dependent on intracellular calcium elevations. J Physiol 561, 499–513. 8. Button, B., Picher, M., and Boucher, R. C. (2007) Differential effects of cyclic and constant stress on ATP release and mucociliary transport by human airway epithelia. J Physiol 580, 577–592.

9. Kreda, S. M., Seminario-Vidal, L., Heusden, C. V., and Lazarowski, E. R. (2008) Thrombin-promoted release of UDP-glucose from human astrocytoma cells. Br J Pharmacol 153, 1528–1537 10. Joseph, S. M., Buchakjian, M. R., and Dubyak, G. R. (2003) Colocalization of ATP release sites and ecto-ATPase activity at the extracellular surface of human astrocytes. J Biol Chem 278, 23331–23342. 11. Kreda, S. M., Okada, S. F., van Heusden, C. A., O’Neal, W., Gabriel, S., Abdullah, L., Davis, C. W., Boucher, R. C., and Lazarowski, E. R. (2007) Coordinated release of nucleotides and mucin from human airway epithelial Calu-3 cells. J Physiol 584, 245–259. 12. Beigi, R., Kobatake, E., Aizawa, M., and Dubyak, G. R. (1999) Detection of local ATP release from activated platelets using cell surface-attached firefly luciferase. Am J Physiol 276, C267–C278. 13. Lundin, A. (2000) Use of firefly luciferase in ATP-related assays of biomass, enzymes, and metabolites. Methods Enzymol 305, 346–370. 14. Taylor, A. L., Kudlow, B. A., Marrs, K. L., Gruenert, D. C., Guggino, W. B., and Schwiebert, E. M. (1998) Bioluminescence detection of ATP release mechanisms in epithelia. Am J Physiol 275, C1391–C1406. 15. DeLuca, M., and McElroy, W. D. (1978) Purification and properties of firefly luciferase. In Methods Enzymol 57, 3–15.

Chapter 4 High-Throughput Quantitative Bioluminescence Imaging for Assessing Tumor Burden Angelina Contero, Edmond Richer, Ana Gondim, and Ralph P. Mason Abstract Bioluminescence imaging (BLI) has emerged during the past 5 years as the preeminent method for rapid, cheap, facile screening of tumor growth and spread in mice. Both subcutaneous and orthotopic tumor models are readily observed with high sensitivity and reproducibility. User-friendly commercial instruments exist and, increasingly, luciferase-expressing tumor cells are available in academic institutions or commercially. There is an increasing literature on routine use of BLI for assessing chemotherapeutic efficacy, drug combinations, dosing, and timing. In addition, BLI may be applied to more sophisticated questions of molecular biology by including specific promoter sequences. This chapter will describe routine methods used to support multiple investigators in our small animal imaging resource. Key words: Luciferase, luciferin, charged-coupled device cameras (CCD), IGOR Pro, bioluminescence.

1. Introduction The concept of bioluminescence to study biochemistry has been around for many years, for example, as the basis for quantifying ATP in snap-frozen histological specimens or tissue extracts (1, 2). However, in vivo application has been spearheaded by Contag et al. (3) and promoted by Xenogen (now Caliper Lifesciences). In less than a decade, BLI has become a routine modality for use in cancer biology, particularly suited for assessing tumor burden and metastatic spread. In vivo BLI has been reviewed many times (3–6), and readers are directed to these papers and other chapters of this book for further insight.

P.B. Rich, C. Douillet (eds.), Bioluminescence, Methods in Molecular Biology 574, DOI 10.1007/978-1-60327-321-3_4, ª Humana Press, a part of Springer Science+Business Media, LLC 2009

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In its most popular format, the bioluminescent reaction requires luciferase enzyme derived from the American firefly (Photinus pyralis) and D-luciferin substrate. Luciferase is generated by cells following transfection. It is important to select clones with high-stable expression, usually based on lentiviral transfection, which tends to be more stable than plasmid transfection. It is important to recognize that clones isolated for high expression may not behave identically to parallel lines or the parental system (e.g., differential growth rates). Thus, tumor models can be highly effective in terms of assessing tumor development and response to therapy, but they may not perfectly replicate parental cell lines. Pharmacokinetics of the luciferin substrate is important. Remarkably, luciferin appears to readily permeate every tissue, including crossing the blood–brain and blood–placental barriers (4). However, the kinetics of light emission can differ with tumor location, and thus it is critical to establish reproducibility of lightemission curves prior to embarking on large-scale studies. The most popular route of administration of luciferin is intra peritoneal (IP) (7); but while this is apparently facile, we find a significant failure rate (8) where no light emission is observed following substrate administration, yet if repeated 1 h later gives expected bioluminescence. We attribute this to poor injection, possibly into the intestines. Intravenous (IV) administration can give much higher light emission (9), but more transiently so that any variation in the timing of image capture and/or integration time can generate poorer reproducibility (8). Intravenous injection is also technically more challenging. Direct intratumor (IT) injection generates the most intense bioluminescence, but is obviously invasive and feasible only for easily accessible tumors (7, 10). We favor subcutaneous (SC) administration of luciferin in the back neck region. The technique is facile with overwhelming success in observing expected signal, and the kinetics provide intense light over several minutes (8, 11). Light detection is strongest from subcutaneous tumor sites, although in this case caliper measurements may be just as effective and cheaper for simple tumor volume assessment. However, BLI is particularly effective for low tumor burdens, and indeed, subpalpable volumes can be detected and quantified (Fig. 4.1). For large tumors, self-absorption and scatter of light can bias apparent relative tumor volume. Planar BLI appears to accurately reflect the volume of small tumors, but becomes less linear for larger tumors although continuing to increase monotonically (12, 13). Light is subject to significant absorption and scattering from deep tumors, and thus equivalent tumors located at depth are expected to provide much less detectable light. Thus, for longitudinal studies, it is crucial to view an animal from the same direction on successive occasions to ensure a reproducible, solid viewing angle and consistent absorption by any intervening tissues. Nude mice are

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Day 7

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Control No cisplastin 106 cells day 0

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Cisplatin 3mg/kg Day 3, 7, 10

Fig. 4.1. Assessing chemotherapy by bioluminescence imaging (BLI). Series of images of two nude mice imaged at various times after injecting HeLa-luc cells into the peritoneum. On each occasion, mice were anesthetized and 150 mg/kg luciferin was administered, followed by a 10-min image. The upper mouse served as control, while the lower series of images show a mouse which received cis-platinum chemotherapy at 3 mg/kg on days 3, 7, and 10, following introduction of tumor cells. Note images have been scaled differently to accommodate the massive changes in dynamic range.

preferred, though light may also be detected from white or black mice with hair: some investigators prefer to shave the animals or apply depilating agents. BLI systems can be constructed quite easily and cheaply based on several recipes in the literature, primarily from the amateur astronomy field, where there is a similar need to detect weak signals against a low background based on longterm signal integration (14). To date, our BLI service uses a home-built system, which has been described elsewhere (7, 8, 15). The primary protocol below describes the procedures with this system (Cyclops). However, the instrument is technically complex, requiring a BLI technician and engineering support. Sophisticated commercial systems are available, which are user-friendly (Caliper Xenogen and Berthtold, see Notes 1 and 2, respectively), and we have recently acquired both IVIS1 Lumina and Spectrum systems for use by multiple research teams. These provide both bioluminescence and fluorescence imaging, including depth-resolved capabilities for the spectrum. D-Luciferin can cost $100 per 100 mg, but bulk purchases should allow better than $400 per gram, which is important for high-throughput screening. Although BLI is simple, several properties require consideration. The light emission can by characterized by parameters including area under the curve (AUC), maximum signal intensity, time to maximum intensity, or light integration over a specified period. We routinely use a dose of 450 mg/kg administered subcutaneously into an anesthetized nude

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mouse, with imaging for a period of 5 min starting 10 min after luciferin administration. Weak signals may require longer integration to achieve useful signal to noise, but many investigators prefer a constant acquisition method even though small tumors then provide essentially zero signal.

2. Materials 2.1. Preparation of Luciferin

1.

D-Luciferin Firefly, sodium salt monohydrate (synthetic) (Biosynth, Staad, Switzerland, See Note 3) stored in dark at –20C.

2. Sorensen’s phosphate buffer, 0.2 M, pH 7.2 (phosphate mixed solution salts) stored at 2–8C (Electron Microscopy Sciences, Hatfield, PA). 2.2. Administration of Luciferin

1. 1/2 cc 28G1/2, U-100 insulin syringes, latex-free syringe, micro-fine IV 2. Veterinary isoflurane, USP 3. USP medical-grade oxygen compressed USP, 99% pure.

2.3. Imaging Equipment

1. A CCD (SITe SI-032AB) non-color, back-illuminated, fullframe image sensor with 512  512 pixels (Scientific Imaging Technologies Inc., Tigard, OR), see Note 4. 2. 1400 Duo-Seal vacuum pump (Welch, Niles, IL). 3. FP88-HL ultra-low refrigerated circulator (Julabo, Allentown, PA). 4. Dark box to accommodate imaging system. 5. Dehumidifier. 6. NIST-traceable research radiometer (IL 1700, International Light, Inc., Newburyport, MA) for camera calibration. 7. Deltaphase isothermal warming pad. 8. Anesthesia system Matrix Medical Inc. (VMC model 100 F, Orchard Park, NY). 9. Data acquisition and processing computer running IGOR Pro (Wavemetrics, Seattle, WA, USA), and custom image analysis routines or Caliper Xenogen IVIS1 Lumina with the LivingImage software (Hopkinton, MA).

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3. Methods 3.1. Preparation of Luciferin

1. Luciferin is used at 40 mg/ml of D-Luciferin sodium salt monohydrate for both mouse and rat studies. 2. Mix the substrate with 2 M Sorensen’s phosphate buffer solution. 3. After making the solution, cover the vial (i.e., foil), since the luciferin is light-sensitive and stored in a laboratory refrigerator.

3.2. Injection of Luciferin

1. Anesthetize the mouse with an induction dose of isoflurane 2.5% in oxygen (see Note 5). 2. Inject a dose of 450 mg/kg luciferin (280 ml for 25 g mouse) subcutaneously in the back neck region using a single-use insulin syringe 10 min prior to imaging (see Note 6). It is important that the time to start imaging is kept constant between individuals and for repeat measurements, since the light emitted is strongly time-dependent. 3. Depending on the region of interest, place the animal on a secure netted bed and place the snout in an anesthesia nose cone, maintained with 1.5% isoflurane and 1 l/min oxygen in the imaging box.

3.3. Imaging with Cyclops (Advanced Radiological Sciences Imaging System)

1. The vacuum pump must be turned on followed by the coolant pump of the refrigerated circulation system. 2. After the refrigerant has cooled to –1.0C, turn on the charge-coupled cameras (CCD) and cool to 230 K (–43C) using internal thermoelectric device. 3. The door of the light box is closed. Prior to imaging the animal, a dark image is acquired to allow subtraction of dark current signal and interference noise from auxiliary equipment. The dark image integration time should be the same as the BLI time, which depends on how intense is the BLI signal. During dark image acquisition, a mechanical shutter shields the camera sensor and therefore an image is taken without any light. 4. Apply diffuse light sources and acquire a 700-ms light image for image overlay to show the body of the animal for orientation and anatomical co-registration. 5. A 5-min BLI image is taken immediately after the light image (see Note 7). In some cases, multiple sequential images are acquired to reveal light-emission dynamics. In high-throughput mode, usually a single image is captured for each animal.

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6. At the end of the imaging series a second dark image is acquired. 7. Return mice to cage and monitor until fully recovered from anesthesia, usually within 5 min. 8. Between groups of mice, spray the animal bed and support structure with Quatricide disinfectant (Pharmacal Research Laboratories Inc., Waterbury, CT) to reduce risk of pathogen spread (Fig. 4.2).

a

b

c

d

Fig. 4.2. Images associated with bioluminescence imaging (BLI). Images acquired with home-built Cyclops BLI system. (a) Dark image to allow subtraction of background noise; (b) light image based on surface external illumination for anatomical registration; (c) bioluminescent image; (d) overlay of bioluminescent signal intensity on anatomical image.

3.3.1. Processing Images

1. Save the images on a personal computer and process using IGOR Pro software with a set of custom image analysis routines (see Note 8). 2. Upload the Dark Image 1, then the Dark Image 2, and average them to allow subtraction of background/instrument noise.

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3. Upload the BLI image and subtract averaged dark image. 4. Create a region of interest (ROI) to measure and integrate the signal. Signals are either measured in relative light units (RLUs) per second, radiance units photons/s/cm2/sr, or total light-emission photons/s. 5. Upload the light image and overlay the bioluminescence image after the background is rendered transparent. 6. Save images as JPG or TIFF files. 3.3.2. Measuring the Signal

1. Using IGOR Pro access ROI under the Image Tools. 2. Draw an ROI around the signal. The signal is measured by creating a box around the signal and integrating the region of interest. 3. Save the data in an Excel file and the picture in your computer drive as JPEG or TIFF files.

3.4. Xenogen Lumina IVIS System ( See Note 9)

The lumina system includes anesthesia unit and heated platform and can image up to three mice at a time. 1. To begin, the user must initiate IVIS system to begin the cooling down process. The optimal time between injection and imaging depends on the route of luciferin injection and tumor site. 2. When ready to image, place the mouse in the anesthesia induction chamber. After turning on the oxygen, the gas valve on the anesthesia is opened. 3. Turn on the evacuation pump before opening the induction chamber. 4. To image the mouse, place the mouse on the platform and open the anesthesia flow for the IVIS box. The door must be closed in order to begin imaging. For multiple mice, a black shield is placed between animals to reduce cross-illumination. 5. There are four fields of view ranging from A (the closest in length to the cameras) to D (the farthest). 6. The user must select bioluminescence since the lumina has both fluorescent and bioluminescent capabilities. Exposure time is set in minutes, and the pixel binning is usually set at medium. 7. After acquiring the image, the mouse must be taken out of the box, and the oxygen tank, anesthesia unit, and gas valve must be turned off. 8. The system remains on overnight to allow the dark images to be taken and saved. This is done automatically.

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9. Save the images on a personal computer and analyze them using the Living Image Data Software provided by Caliper. 10. The lumina system automatically corrects light intensity for camera–subject distance and calibrates for dark images daily.

4. Notes 1. http://www.caliperls.com/products/ivis-lumina.htm 2. http://www.berthold.com/bio/ww/en/pub/bioanalytik/ produkte/lb981.cfm 3.

D-Luciferin

may be obtained from many sources as either synthetic or natural material. Sodium or potassium salts may be used. We have no evidence for differential quality. Other sources include D-luciferin sodium salt (Catalog #10102; Biotium, Hayward, CA, USA); D-luciferin potassium salt (P/N 122769) isolated from firefly (Caliper Life Sciences: http://www.caliperls.com/products/dluciferin-potassiumsalt.htm).

4. In earlier work we had used a TC245 CCD camera (Texas Instruments, Dallas TX) (7) and a system based on the French Audine astronomical camera with a high-performance Kodak KAF-0402ME CCD (7). 5. Other forms of anesthesia, such as ketamine, can also be used. 6. Other doses of luciferin may be used. Caliper recommends a dose of 150 mg/kg for mice with its lumina imaging system. Our experience favors the higher dose. Caliper Lifesciences recommends 10–15 min between injection and imaging. 7. The exposure time may be altered to avoid overexposing intense signals or to detect weak signals. In practice, we may use anywhere from 1 to 30 min. Many investigators like to maintain a constant imaging time, where 5 min is typical. 8. The macros are really outside the scope of this chapter and interested readers are referred to (8). 9. Instructions derived from the instrument user manual.

Acknowledgments Supported in part by grants from the DOD Breast Cancer Initiative (IDEA award DAMD17-03-1-0343), the NIH Cancer Imaging Program (P20 CA86354 and U24 CA126608), and the

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Simmons Cancer Center. We are grateful to Drs. Li Liu, Robert Bollinger, Jerry Shay, and Peter Antich for bringing the vision of BLI to UT Southwestern. References 1. Schaefer, C., Mayer, W. K., Kru ¨ ger, W., and Vaupel, P. (1993) Microregional distributions of glucose, lactate, ATP and tissue pH in experimental tumours upon local hyperthermia and/or hyperglycaemia. J Cancer Res Clin Oncol 119, 599–608. 2. Lundin, A. (2000) In Bioluminescence and Chemiluminescence, Pt C 305 346–370. 3. Contag, C. H., and Ross, B. D. (2002) It’s not just about anatomy: in vivo bioluminescence imaging as an eyepiece into biology. JMRI 16, 378–387. 4. Thorne, S. H., and Contag, C. H. (2005) Using in vivo bioluminescence imaging to shed light on cancer biology. Proc IEEE 93, 750–762. 5. Massoud, T. F., and Gambhir, S. S. (2003) Molecular imaging in living subjects: seeing fundamental biological processes in a new light. Genes Dev 17, 545–580. 6. Bhaumik, S., and Gambhir, S. S. (2002) Optical imaging of Renilla luciferase reporter gene expression in living mice. Proc Natl Acad Sci USA 99, 377–382. 7. Paroo, Z., Bollinger, R. A., Braasch, D. A., Richer, E., Corey, D. R., Antich, P. P., and Mason, R. P. (2004) Validating bioluminescence imaging as a high-throughput, quantitative modality for assessing tumor burden. Mol Imaging 3, 117–124. 8. Bollinger, R. A. (2006), Ph.D., UT Southwestern, Dallas. 9. Wang, W., and El-Deiry, W. S. (2003) Bioluminescent molecular imaging of endogenous and exogenous p53-mediated transcription in vitro and in vivo using an HCT116 human

10.

11.

12.

13.

14.

15.

colon carcinoma xenograft model. Cancer Biol Ther 2, 196–202. Cecic, I., Chan, D. A., Sutphin, P., Ray, P., Gambhir, S. S., Giaccia, A. J., and Graves, E. E. (2007) Oxygen sensitivity of reporter genes: implications for preclinical imaging of tumor hypoxia. Mol Imaging Biol 6, 219–228. Karam, J. A., Fan, J., Stanfield, J., Richer, E., Benaim, E. A., Frenkel, E., Antich, P., Sagalowsky, A. I., Mason, R. P., and Hsieh, J. -T. (2007) The use of histone deacetylase inhibitor FK228 and DNA hypomethylation agent 5-Azacytidine in human bladder cancer therapy. Int J Cancer 120, 1795–1802. Klerk, C. P., Overmeer, R. M., Niers, T. M., Versteeg, H. H., Richel, D. J., Buckle, T., Van Noorden, C. J., and van Tellingen, O. (2007) Validity of bioluminescence measurements for noninvasive in vivo imaging of tumor load in small animals. Biotechniques 43, 7–13 Sarraf-Yazdi, S., Mi, J., Dewhirst, M. W., and Clary, B. M. (2004) Use of in vivo bioluminescence imaging to predict hepatic tumor burden in mice. J Surg Res 120, 249–255. Kanto, V., Munger, J., and Berry, R. (1994) The CCD Camera Cookbook, Willman-Bell, Inc, Richmond, VA. Dikmen, Z. G., Gellert, G., Dogan, P., Mason, R., Antich, P., Richer, E., Wright, W. E., and Shay, J. E. (2005) A new diagnostic system in cancer research: bioluminescent imaging (BLI). Turk J Med Sci 35, 65–70.

Chapter 5 Fluorescence Imaging of Tumors with ‘‘Smart’’ pH-Activatable Targeted Probes Daisuke Asanuma, Hisataka Kobayashi, Tetsuo Nagano, and Yasuteru Urano Abstract One goal of molecular imaging is to establish a widely applicable technique for specific detection of tumors with minimal background originated from non-target tissues. In this study, a ‘‘smart’’ activatable strategy for specific tumor imaging is proposed in which pH-activatable targeted probes specifically detect tumors after binding to the target cell surface proteins, internalization, and eventual acidic pH activation within the acidic organelles. We successfully visualized submillimeter-sized tumors using this strategy in two different tumor mouse models. Since the design of pH-activatable targeted probes can be applied to any target molecules on the cell surface that are to be internalized after ligand binding, this imaging strategy can afford a general and powerful method to diagnose and monitor the target tumors. Keywords: Optical tumor imaging, fluorescence, molecular probes, pH, receptor-mediated endocytosis.

1. Introduction Molecular imaging has been efficaciously employed to detect and guide treatment of tumors (1, 2). Accurate diagnosis of lesions is crucial for the success of cancer therapy, including cytoreduction and surgical metastasectomy. Recently, fluorescence imaging techniques have attracted interest in clinical oncology because of its high sensitivity and specificity, excellent temporal and spatial resolution, and low-cost imaging systems without ionizing radiation, relative to other imaging modalities such as PET/SPECT, MRI, and CT. The feasibility of tumor imaging depends on how to distinguish between lesions and normal sites in pathophysiological aspects and to specifically target tumors with some distinctive features. Overexpressingcellsurfaceproteinsontumors,suchassomatostatinreceptor(3)or folate receptor (4), are excellent candidates for targeted tumor imaging. P.B. Rich, C. Douillet (eds.), Bioluminescence, Methods in Molecular Biology 574, DOI 10.1007/978-1-60327-321-3_5, ª Humana Press, a part of Springer Science+Business Media, LLC 2009

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In conventional approaches of tumor imaging with fluorophore-conjugated peptides or antibodies targeted to these cell surface markers, however, one of the problems is a limited tumor-to-background ratio because of the ‘‘always on’’ nature of the imaging probes regardless of their distribution in tumors, normal tissues, or blood (Fig. 5.1A). For specific tumor imaging, ‘‘smart’’ activatable probes have been developed, which increase their fluorescence intensity after target reaction (5–8) (Fig. 5.1B). Because of their low initial fluorescence and targeted activation,highertumor-to-backgroundratioscanbeachievedthanthe above-mentioned approaches.

Fig. 5.1. Mechanism of a cancer imaging strategy using target-specific activatable probes. (A) Conventional strategies for tumor imaging with MRI, PET, or nonactivatable ‘‘always on’’ fluorescence detection. (B) New strategy for selective tumor imaging with activatable fluorescence probes. (C) A schematic representation of highly selective tumor imaging with pH-activatable targeted probes. The probe is nonfluorescent when outside the tumor cells. After internalization by endocytosis, the probe is accumulated in late endosomes or lysosomes, where the acidic pH activates the probe, making it highly fluorescent. S/N: Signal/Noise ratio.

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49

Our strategy for specific tumor imaging exploits ligandinduced internalization and subsequent delivery of cell surface protein–ligand complexes to the acidic organelles such as late endosome and lysosome, leading to acidic pH activation of fluorescence probes tethered to the ligands (Fig. 5.1C). Several tumorassociated cell surface markers have been reported demonstrating such properties: b-D-galactose receptor (lectin) (9), human epidermal growth factor receptor type 2 (HER2) (10), transferrin receptor (11), LDL receptor (12), membrane type 1-matrix metalloprotease (MT1-MMP) (13), and so on. Labeling of ligands targeted to these markers with our developed, acidic pH-activatable fluorescence probes (Fig. 5.2) can afford pH-activatable targeted probes, which remain ‘‘silent’’ in the extracellular environment in vivo at physiological pH, but turn ‘‘on’’ only after specific internalization into the target tumor cells (Fig. 5.1C).

A R2

R1

Switch moiety

R2

N

R1

Fluorophore

O HO

N F

B

N F

H O OH

–H+

O

Switch moiety NH 2

pH Activatable probes

H2NBDP N

DiMeNBDP N

EtMeNBDP N

Control

DiEtNBDP

PhBDP

N

HO

Almost non-fluorescent

B

NH

+

F

B

O

N F

OH

Highly fluorescent (Ex/Em = 520/537 nm)

C Fluorescence quantum yield

e–

0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

0

2

4

6

8

10

pH

H2NBDP (pKa = 3.8) DiMeNBDP (pKa = 4.3) EtMeNBDP (pKa = 5.2) DiEtNBDP (pKa = 6.0) PhBDP (always on)

Fig. 5.2. Development of a series of fluorescence probes for various acidic environments. (A) A scheme for the reversible and acidic pH-induced fluorescence activation of probes. (B) pH profiles of fluorescence of H2NBDP, DiMeNBDP, EtMeNBDP, and DiEtNBDP as acidic pH-sensitive fluorescence probes and PhBDP as a control ‘‘always on’’ probe. The pH ranges from 2 to 9 in one pH unit increments. (C) pH-dependent changes in emission intensity of acidic pH-activatable probes. Curve fitting was based on Henderson–Hasselbach equation. Fluorescence quantum yield (fl) of the probes versus pH, measured in 200 mM sodium phosphate buffer and determined with fluorescein (fl ¼ 0.85 in 0.1 N NaOH aq.) as a standard.

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We demonstrate that, after laparotomy or thoracotomy, it is possible to detect submillimeter-sized tumors using this strategy in two different tumor mouse models. One is a lectin-overexpressing model, which can be targeted with galactosamine-conjugated serum albumin (GSA), while the other is a HER2-overexpressing one with Herceptin, a monoclonal antibody against HER2. For comparison, ‘‘always on’’ PhBDP (Fig. 5.1B, C) was also employed for tumor imaging as a control, indicating importance of the activatable strategy for specific tumor imaging.

2. Materials 2.1. Synthesis of pH-Activatable and Non-activatable Fluorescence Probes

Materials for the synthesis of the probes: 1,3,5,7-Tetramethyl-2,6bis-(2-carboxyethyl)-8-[4-(N,N-diethylamino)phenyl]-4,4-difluoro4-bora-3a, 4a-diaza-s-indacene (DiEtNBDP) and 1,3,5,7-tetramethyl-2,6-bis-(2-carboxyethyl)-8-phenyl-4,4-difluoro-4-bora3a,4a-diaza-s-indacene (PhBDP), and their mono-succinimidyl esters (DiEtNBDP, SE&PhBDP, SE) 1. Methyl 5-(benzyloxycarbonyl)-2,4-dimethyl-3-pyrrolepropionate 2. 4-(N,N-Diethylamino)benzaldehyde (for DiEtNBDP) 3. Benzaldehyde (for PhBDP) 4. Trifluoroacetic acid (TFA) 5. 10% Palladium-carbon 6. 2,3,5,6-Tetrachloro-1,4-benzoquinone (p-chloranil) 7. N,N-Diisopropylethylamine 8. Boron trifluoride etherate 9. Sodium hydroxide 10. Acetone 11. Dichloromethane 12. Methanol 13. Toluene

2.2. Preparation of pH-Activatable and Non-activatable Targeted Probes

1. Albumin, bovine-galactosamide (23 mol galactosamine/mol albumin) (galactosamine-conjugated serum albumin: GSA) 2. Herceptin1 (Genentech Inc., South San Francisco, CA) 3. Dimethyl sulfoxide (DMSO) 4. PD-10 columns (SephadexTM G-25 M) (GE Healthcare, Poole, UK) 5. PBS pH 7.4

2.3. Cell Culture

1. RPMI 1640 medium 2. Fetal bovine serum (FBS)

Fluorescence Imaging of Tumors

3. Penicillin/streptomycin: 10,000 10,000 mg/mL streptomycin

units/mL

51

penicillin,

4. Trypsin–EDTA: 0.05% trypsin, 0.53 mM EDTA-4Na 2.4. Injection of Tumor Cells and Targeted Probes into Mouse Models

1. Tuberculin syringes (1 cc)

2.5. Imaging System

1. MaestroTM In-Vivo Imaging System (CRi Inc., Woburn, MA)

2. 26G  1/200 needles (0.45  13 mm)

3. Methods 3.1. Imaging of Tumors with GSA-DiEtNBDP in Tumor Mouse Models 3.1.1. Synthesis of pH-Activatable and Non-activatable Fluorescence Probes O

O O

O H2 /10%Pd-C

O

acetone, rt

N H

O

TFA N H

1 y. 94% (2 steps) R 1) 1 (2 equiv.) DIEA cat. TFA BF3 .OEt2 CH 2Cl2, rt

R

2) p-chloranil toluene, rt rt O

O

H

O

R = NEt2 R= H

N

B

F

O

N F

O

2a R = NEt2 y. 16% y. 32% 2b R = H R

R

NaOH CH 2Cl2 /MeOH/H2 O rt

NHS, WSCD DMF, 0oC to rt

O HO

N F

B

N F

O OH

DiEtNBDP (3a) R = NEt2 y. 91% y. 75% PhBDP (3b) R = H

O HO

N F

B

N F

O

O

O N

DiEtNBDP, SE (4a) R = NEt2 y. 44% O PhBDP, SE (4b) R = H y. 27%

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3.1.1.1. Methyl 2,4dimethyl-3pyrrolepropionate

Methyl 5-(benzyloxycarbonyl)-2,4-dimethyl-3-pyrrolepropionate (1.55 g, 4.91 mmol) is dissolved in 150 mL of acetone containing 10% palladium-carbon. The resulting solution is stirred under H2 at ambient temperature (rt: room temperature) for 12 h. The reaction solution is then filtered and evaporated. The residue is immediately dissolved in 10 mL of TFA and stirred under an argon atmosphere at ambient temperature for 10 min. Then, 30 mL of dichloromethane is added and the resulting solution is washed with H2O and 1 M NaHCO3 aq., dried over anhydrous sodium sulfate, filtered, and evaporated, resulting in 1 (0.835 g, 94%) as a slightly brown oil. 1H NMR (300 MHz, CDCl3) d 2.02 (s, 3H, NHCHCCH3), 2.16 (s, 3H, NHCCH3), 2.42–2.48 (m, 2H, COCH2), 2.69–2.74 (m, 2H, COCH2CH2), 3.66 (s, 3H, OCH3), 6.36 (s, 1H, NHCH), 7.64 (br s, 1H, NH); 13C NMR (75 MHz, CDCl3) d 10.22, 11.09, 19.85, 35.26, 51.37, 113.0, 116.5, 117.7, 124.1, 173.9. MS (ESI+) m/z 182 [M+H]+.

3.1.1.2. 1,3,5,7Tetramethyl-2,6-bis(2-methoxycarbonylethyl)8-[4-(N,Ndiethylamino)phenyl]4,4-difluoro-4-bora3a,4a-diaza-s-indacene (2a)

1 (0.542 g, 2.99 mmol) and 4-(N,N-diethylamino)benzaldehyde (0.265 g, 1.49 mmol) are dissolved in 300 mL of dichloromethane containing a catalytic amount of TFA. The resulting mixture is stirred overnight at ambient temperature under an argon atmosphere. p-Chloranil (0.370 g, 1.51 mmol) is added, and stirring is continued for 10 min. The reaction mixture is washed with H2O, dried over anhydrous sodium sulfate, filtered, and evaporated. Repeated column chromatography over aluminum oxide using dichloromethane/methanol (95:5, 98:2, and 100:0) containing 1% triethylamine as the eluent yields a greenish amorphous compound. The compound thus obtained is dissolved in 100 mL of toluene containing DIEA (5 mL), and the resulting solution is stirred at ambient temperature. BF3OEt2 (5 mL) is then slowly added, and stirring is continued for 10 min. The reaction mixture is washed with H2O, dried over anhydrous sodium sulfate, filtered, and evaporated. The crude compound is purified by repeated column chromatography over silica gel using dichloromethane/ methanol (95:5, 98:2, and 100:0) as the eluent, resulting in 2a (136 mg, 16%) as an orange powder. 1H NMR (300 MHz, CDCl3) d 1.22 (t, 6H, J ¼ 7.0 Hz, NCH2CH3), 1.44 (s, 6H, NCCCH3), 2.36 (t, 4H, J ¼ 7.3, 8.4 Hz, COCH2), 2.53 (s, 6H, NCCH3), 2.65 (t, 4H, J ¼ 7.3, 8.4 Hz, COCH2CH2), 3.41 (q, 4H, J ¼ 7.0 Hz, NCH2), 3.65 (s, 6H, OCH3), 6.74 (d, 2H, J ¼ 8.6 Hz, NCCHCH), 6.99 (d, 2H, J ¼ 8.6 Hz, NCCH); 13C NMR (75 MHz, CDCl3) d 12.11, 12.30, 12.45, 19.34, 34.25, 44.31, 51.56, 112.0, 121.6, 128.6, 129.0, 131.7, 139.6, 142.6, 148.2, 153.1, 173.1; HRMS (ESI+) calculated value for [M+H]+ m/z 568.31582, found 568.31626 (D 0.44 mmu).

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3.1.1.3. 1,3,5,7Tetramethyl-2,6-bis-(2carboxyethyl)-8-[4-(N,Ndiethylamino)phenyl]-4,4difluoro-4-bora-3a,4adiaza-s-indacene (3a)

2a (136 mg, 239 mmol) is dissolved in 3 mL of dichloromethane. To the resulting solution are added 20 mL of methanol and 5 mL of 1 N NaOH aq., successively. The reaction solution is stirred for 4 h at ambient temperature. Then 30 mL of H2O is added, and the reaction solution is washed with dichloromethane three times. The aqueous phase is then acidified with 1 N HCl aq. (1 mL) until the solution emits green fluorescence on UV excitation of 365 nm, followed by extraction with dichloromethane three times. The dichloromethane extract is dried over anhydrous sodium sulfate, filtered, and evaporated. The crude compound is then purified by PLC using dichloromethane/acetone (1:1) as the eluent, affording 3a (118 mg, 91%) as an orange powder. 1H NMR (300 MHz, CD3OD) d 1.10 (t, 6H, J¼7.0 Hz, NCH2CH3), 1.39 (t, 6H, NCCCH3), 2.25 (t, 4H, J¼7.5, 7.9 Hz, COCH2), 2.39 (s, 6H, NCCH3), 2.56 (t, 4H, J ¼ 7.5, 7.9 Hz, COCH2CH2), 3.33 (q, 4H, J¼7.0 Hz, NCH2), 6.75 (d, 2H, J ¼ 8.8 Hz, NCCHCH), 6.93 (d, 2H, J ¼ 8.8 Hz, NCCH); 13C NMR (75 MHz, CD3OD) d 12.47, 12.66, 20.38, 35.31, 45.40, 113.3, 122.8, 130.3 (representing two different carbons), 132.8, 140.8, 144.2, 149.7, 154.4, 176.5; HRMS (ESI–) calculated value for [M–H]– m/z 538.26887, found 538.26446 (D–4.40 mmu).

3.1.1.4. 1,3,5,7Tetramethyl-2-(2carboxyethyl)-6(succinimidyl oxycarbonylethyl)-8-[4(N,N-diethylamino)phenyl]-4,4-difluoro-4bora-3a,4a-diaza-sindacene (4a)

3a (25.7 mg, 47.6 mmol) is dissolved in 2 mL of N,N-dimethylformamide (DMF) and the resulting solution is cooled to 0C. To the reaction solution are added 100 mM NHS in DMF and 100 mM WSCD in DMF (each 47.6 mmol). The reaction mixture is stirred at 0C and then allowed to warm gradually to ambient temperature. After 14 h, the reaction mixture is concentrated in vacuo. The crude compound is purified by PLC using dichloromethane/acetone (1:1) as the eluent, resulting in 4a (13.4 mg, 44%) as a red powder. HRMS (ESI+) calculated value for [M+H]+ m/z 637.30090, found 637.30278 (D 1.89 mmu).

3.1.1.5. 1,3,5,7Tetramethyl-2,6-bis-(2methoxycarbonylethyl)-8phenyl-4,4-difluoro-4bora-3a,4a-diaza-sindacene (2b)

1 (0.634 g, 3.50 mmol) and benzaldehyde (0.185 g, 1.74 mmol) are dissolved in 300 mL of dichloromethane containing a catalytic amount of TFA. The resulting mixture is stirred overnight at ambient temperature under an argon atmosphere. p-Chloranil (0.428 g, 1.74 mmol) is added, and stirring is continued for 10 min. The reaction mixture is washed with H2O, dried over anhydrous sodium sulfate, filtered, and evaporated. Repeated column chromatography over aluminum oxide using dichloromethane containing 1% triethylamine as the eluent yields a brown oil. The compound thus obtained is dissolved in 100 mL of toluene containing DIEA (5 mL), and the resulting solution is stirred at ambient temperature. BF3OEt2 (5 mL) is then slowly added, and stirring is continued for 10 min. The reaction mixture is washed with H2O, dried over anhydrous sodium sulfate, filtered,

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and evaporated. The crude compound is purified by column chromatography over silica gel using dichloromethane as the eluent, affording 2b (273 mg, 32%) as a green compound. 1H NMR (300 MHz, CDCl3) d 1.29 (s, 6H, NCCCH3), 2.32–2.38 (m, 4H, COCH2), 2.54 (s, 6H, NCCH3), 2.61–2.66 (m, 4H, COCH2CH2), 3.65 (s, 6H, OCH3), 7.25–7.28 (m, 2H, benzene), 7.46–7.49 (m, 3H, benzene); 13C NMR (75 MHz, CDCl3) d 11.77, 12.59, 19.29, 34.18, 51.63, 128.0, 128.9, 129.1, 130.9, 135.4, 139.4, 140.9, 154.0, 173.0; HRMS (ESI+) calculated value for [M+Na]+ m/z 519.22426, found 519.22433 (D 0.07 mmu). 3.1.1.6. 1,3,5,7Tetramethyl-2,6-bis-(2carboxyethyl)-8-phenyl4,4-difluoro-4-bora-3a,4adiaza-s-indacene (3b)

2b (40.1 mg, 78.4 mmol) is dissolved in 1 mL of dichloromethane. To the resulting solution are added 20 mL of methanol and 5 mL of 1 N NaOH aq., successively. The reaction solution is stirred overnight at ambient temperature. Then 30 mL of H2O is added, and the reaction solution is washed with dichloromethane three times. The aqueous phase is then acidified with 1 N HCl aq. (1 mL) until the solution emits green fluorescence on UV excitation of 365 nm, followed by extraction with dichloromethane five times. The dichloromethane extract is dried over anhydrous sodium sulfate, filtered, and evaporated. The crude compound is then purified twice by semi-preparative HPLC under the following conditions: A/B = 50/50 (0 min)–0/100 (20 min), then A/B ¼ 70/ 30 (0 min)–0/100 (30 min) (solvent A: H2O, 0.1% TFA; solvent B: acetonitrile/H2O = 80/20, 0.1% TFA). The aqueous fractions containing the desired product are extracted with dichloromethane three times. The dichloromethane extract is dried over anhydrous sodium sulfate, filtered, and evaporated, affording 3b (32.0 mg, 84%) as an orange powder. 1H NMR (300 MHz, CD3OD) d 1.19 (s, 6H, NCCCH3), 2.23 (t, 4H, J ¼ 8.1 Hz, COCH2), 2.40 (s, 6H, NCCH3), 2.55 (t, 4H, J ¼ 8.1 Hz, COCH2CH2), 7.21–7.46 (m, 5H, benzene); 13C NMR (75 MHz, CD3OD/NaOD) d 12.19 (representing two different carbons), 22.01, 39.34, 129.5, 130.2, 130.4, 132.0, 132.2, 136.9, 140.4, 142.1, 155.2, 181.8; HRMS (ESI+) calculated value for [M+Na]+ m/z 491.19296, found 491.18910 (D –3.87 mmu).

3.1.1.7. 1,3,5,7Tetramethyl-2-(2carboxyethyl)-6(succinimidyl oxycarbonylethyl)-8phenyl-4,4-difluoro-4bora-3a,4a-diaza-sindacene (4b)

3b (12.4 mg, 26.5 mmol) is dissolved in 1 mL of N,N-dimethylformamide (DMF) and the resulting solution is cooled to 0C. To the reaction solution are added 100 mM NHS in DMF and 100 mM WSCD in DMF (each 39.7 mmol). The reaction mixture is stirred at 0C and then allowed to warm gradually to ambient temperature. After 24 h, the reaction mixture is concentrated in vacuo. The crude compound is then purified by semi-preparative HPLC under the following conditions: A/B = 50/50 (0 min)– 0/100 (20 min) (solvent A: H2O, 0.1% TFA; solvent B:

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55

acetonitrile/H2O = 80/20, 0.1% TFA). The aqueous fractions containing the desired product are extracted with dichloromethane three times. The dichloromethane extract is dried over anhydrous sodium sulfate, filtered, and evaporated, affording 4b (4.0 mg, 27%) as a red powder. Recovery is 24%. HRMS (ESI–) calculated value for [M–H]– m/z 564.21175, found 564.21392 (D 2.18 mmu). 3.1.2. Labeling of GSA with DiEtNBDP or PhBDP

Synthetic schemes of pH-activatable fluorescence probes are also referred in (14). Succinimidyl esters (SEs) are excellent reagents for protein labeling because of their high reactivity with primary amines, such as lysine residues, to form stable amide bonds. A carboxylic group of DiEtNBDP or PhBDP is readily converted to a succinimidyl ester by condensation reaction with N-hydroxysuccinimide, providing DiEtNBDP, SE and PhBDP, SE, respectively. 1. Dissolve each DiEtNBDP, SE and PhBDP, SE in DMSO to afford 10 mM stock solutions (see Note 1). 2. Dissolve GSA in 200 mM sodium phosphate buffer (pH 8.5) (see Note 2) to obtain 1.0 mg/mL GSA stock solution (14.2 nmol/mL). 3. Add 22.7 mL of 10 mM DiEtNBDP, SE stock solution (227 nmol; 16 eq.) or 14.2 mL of 10 mM PhBDP, SE stock solution (142 nmol; 10 eq.) to 1.0 mL of 1.0 mg/mL GSA stock solution (14.2 nmol), immediately followed by gentle mixing. 4. Incubate the reaction solutions for 60 min at ambient temperature in the dark. 5. Separate the GSA-DiEtNBDP and GSA-PhBDP conjugates from free DiEtNBDP and PhBDP, respectively, by PD-10 columns using PBS pH 7.4 as the eluent according to the manufacturer’s instruction, yielding 3.0 mL of stock solution of GSA-DiEtNBDP and GSA-PhBDP. Preserve the stock solution in the dark at 4C (see Note 3).

3.1.3. Determination of the Degree of Labeling (DOL) for GSA-DiEtNBDP and GSAPhBDP

DOL is defined as labeled fluorophore [mol]/protein [mol], which serves as one of the most important indicators of conjugates determining their functions and stability. Less DOL of the conjugates, better their stability and less their functions (e.g., pKa values for pH-activatable probes and Kd values for ligands) affected relative to those of individual molecule before labeling, but less their fluorescence signals a conjugate molecule, resulting in reduced sensitivity in imaging experiments or vice versa. The DOL of conjugates should be optimized in each combination of fluorescence probes and proteins.

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1. Dilute the GSA-DiEtNBDP stock solution (50 mL) with PBS pH 7.4 (2450 mL) in a cuvette (light path length (l) = 1 cm). The absorbance (Abs) of GSA-DiEtNBDP at 520 nm was determined to be 0.03137 with an absorption spectrometer. 2. Calculate the DOL for GSA-DiEtNBDP (DiEtNBDP/GSA [mol/mol]) by using the following equation: DOL ¼

Abs  Dilution 1  ; l c

[1]

where Dilution is a dilution factor used for absorbance measurement, e is molar extinction coefficient (L/mol/ cm) of the labeled probe, and c is protein concentration (mol/L). Assuming that (i) there is no change for e of DiEtNBDP before and after GSA conjugation and (ii) there is no loss of macromolecular GSA at the separation step (1,3,5,7-tetramethyl-2,6-bis-(2-methoxycarbonylethyl) -8-phenyl-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (2b)), e and c were determined to be 64,000 (L/mol/cm) and 4.73  10–6 (mol/L), respectively. The DOL is determined as follows:

DOL ¼

0:0137:50 1  ¼ 5:2: 64; 000:1 4:73  106

[2]

3. Similarly, the DOL was determined to be 4.5 for GSAPhBDP. 3.1.4. Preparation of Mouse Models of Intraperitoneally Disseminated Tumor

1. Culture the human ovarian cancer cell line SHIN3 in RPMI 1640 containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin at 37˚C in humidified air containing 5% CO2. 2. When SHIN3 cells reach sub-confluence, treat the cells with trypsin–EDTA to separate culture into single cells (see Note 4). Just after centrifugation (100 g, 3 min, 4C) of a solution of the trypsinized SHIN3 cells, remove the supernatant and suspend the cells with ice-cold PBS pH 7.4 to a cell density of 1  106 cells/300 mL PBS. 3. Immediately, inject 1  106 cells of SHIN3 suspended in 300 mL of PBS in athymic nude mice (see Note 4). 4. Breed treated mice for 10–14 days when a centimeter-sized tumor is intraperitoneally formed adjacent to the pancreas, and millimeter-sized tumors are disseminated on the mesentery (see Note 5).

Fluorescence Imaging of Tumors

1. Inject 100 mg of GSA-DiEtNBDP (DOL = 5.2) as a pH-activatable targeted probe or 100 mg of GSA-PhBDP (DOL = 4.5) as a control in 300 mL of PBS into the peritoneal cavity of the mouse models of intraperitoneally disseminated tumor. 2. At 2–3 h post-injection, kill the mice by CO2 treatment, followed by whole blood collection. 3. Expose surgically the abdominal cavity of the treated mouse models with scissors and tweezers for small-animal use. 4. Capture the white light and fluorescence spectral images of the whole abdominal cavity and the mesentery with a MaestroTM In-Vivo Imaging System (CRi Inc., Woburn, MA) (Figs. 5.3 and 5.4). The fluorescence emission spectra are obtained from 520 to 800 nm in 10-nm step with excitation at 445–490 nm. 5. Create the unmixed images with the use of authentic spectral patterns of DiEtNBDP, PhBDP, and the background. Figure 5.3C, D show unmixed images of the peritoneal cavity of the treated mouse models, where probe fluorescence was visualized as green, while autofluorescence originated from the skin and the internal duct white and orange, respectively. Moreover, in Fig. 5.4, autofluorescence from the adipose tissue was additionally separated and assigned yellow.

White light image

GSA-PhBDP (always ON)

Unmixed image

3.1.5. Fluorescence Spectral Imaging in the Mouse Models of Intraperitoneally Disseminated Tumor

57

GSA-DiEtNBDP (pH-activatable)

A

B

C

D

Fig. 5.3. The activatable GSA–DiEtNBDP can specifically detect intraperitoneal tumors. White light (A and B) and fluorescence unmixed images (C and D) of the peritoneal cavity in the mouse models of intraperitoneally disseminated tumor with ‘‘always on’’ GSA– PhBDP (A and C) or ‘‘pH-activatable’’ GSA–DiEtNBDP (B and D). Unmixed images indicate probe fluorescence (green) and autofluorescence originated from the skin (white) and the internal duct (orange). White arrowheads indicate disseminated tumors.

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White light image

A

Unmixed image

GSA-PhBDP (always ON)

C

GSA-DiEtNBDP (pH-activatable)

B

5 mm

D

Fig. 5.4. The activatable GSA–DiEtNBDP can specifically detect intraperitoneally disseminated tumors as small as submillimeter in size. White light (A and B) and fluorescence unmixed images (C and D) of the mesentery of the mouse models of intraperitoneally disseminated tumor with ‘‘always on’’ GSA–PhBDP (A and C) or ‘‘pHactivatable’’ GSA–DiEtNBDP (B and D). Unmixed images indicate probe fluorescence (green) and autofluorescence originated from the internal duct (orange) and the adipose tissue (yellow). Scale bar is 5 mm.

3.2. Imaging of Tumors with Herceptin– DiEtNBDP in Tumor Mouse Models 3.2.1. Labeling of Herceptin with DiEtNBDP or PhBDP

1. Dissolve Herceptin in 200 mM sodium phosphate buffer (pH 8.5) (see Note 2) to obtain 1.0 mg/mL Herceptin stock solution (3.42 nmol/mL). 2. Add 3.42 mL of 10 mM DiEtNBDP, SE stock solution (34.2 nmol; 10 eq.) or 2.05 mL of 10 mM PhBDP, SE stock solution (20.5 nmol; 6 eq.) to 1.0 mL of 1.0 mg/mL Herceptin stock solution (3.42 nmol), immediately followed by gentle mixing. 3. Incubate the reaction solutions for 60 min at ambient temperature in the dark. 4. Separate the Herceptin–DiEtNBDP and Herceptin–PhBDP conjugates from free DiEtNBDP and PhBDP, respectively, by PD-10 columns using PBS pH 7.4 as the eluent according to the manufacturer’s instruction, yielding 3.0 mL of stock solution of Herceptin–DiEtNBDP and Herceptin–PhBDP. The stock solution is preserved in the dark at 4C.

3.2.2. Determination of DOL for Herceptin–DiEtNBDP and Herceptin–PhBDP

1. Measure the absorbance of Herceptin–DiEtNBDP and Herceptin–PhBDP at 520 nm with an absorption spectrometer.

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2. Calculate the DOL with equation. In our case, DOL was 2.8 and 3.0 for Herceptin–DiEtNBDP and Herceptin–PhBDP, respectively. 3.2.3. Preparation of Mouse Models of Lung Metastatic Tumor

1. Culture the HER2-transfected NIH3T3/HER2 cells in RPMI 1640 containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin at 37˚C in humidified air containing 5% CO2. 2. When NIH3T3/HER2 cells reach sub-confluence, treat the cells with trypsin–EDTA to separate each cell (see Notes 4 and 6). Just after centrifugation (100 g, 3 min, 4C) of a solution of the trypsinized NIH3T3/HER2 cells, remove the supernatant and suspend the cells to a cell density of 1  106 cells/100 mL with ice-cold PBS pH 7.4. 3. Immediately, inject 2  106 cells of NIH3T3/HER2 suspended in 200 mL of PBS via the tail vein in athymic nude mice (see Note 4). 4. Breed the treated mice for 18–21 days, when millimetersized tumors are formed on the lung surfaces (see Note 5).

3.2.4. Fluorescence Spectral Imaging in the Mouse Models of Lung Metastatic Tumor

1. Inject 300 mg of Herceptin–DiEtNBDP (DOL = 2.8) as a pH-activatable targeted probe, or 100 mg of Herceptin– PhBDP (DOL = 3.0) plus 200 mg of Herceptin as a control via the tail vein into the mouse models of lung metastatic tumor. 2. One day post-injection, kill the mice by CO2 treatment, followed by whole blood collection. 3. Expose surgically the thoracic cavity of the treated mouse models with scissors and tweezers for small-animal use. 4. Capture the white light and fluorescence spectral images of the thorax with a MaestroTM In-Vivo Imaging System (Fig. 5.5). The fluorescence emission spectra are obtained from 520 to 800 nm in 10-nm step with excitation at 445–490 nm. 5. Create the unmixed images with the use of authentic spectral patterns of DiEtNBDP, PhBDP, and the background.

3.3. Conclusion

We successfully visualized tumors as small as submillimeter in size, with minimal background by GSA–DiEtNBDP and Herceptin–DiEtNBDP in the intraperitoneally disseminated tumor and lung metastatic tumor mouse models, respectively. Since the design of pH-activatable targeted probes can be applied to any target molecules on the cell surface that are to be internalized

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Fig. 5.5. The activatable Herceptin–DiEtNBDP can specifically detect submillimetersized tumors on the lung surfaces. (A and D) White light images, (B and E) unmixed images, and (C and F) composite images of (A) and (B), or (D) and (E). The images show simultaneously two operated mice that were arranged side by side with their lungs centered at their position. Left mouse was treated with ‘‘always-ON’’ Herceptin–PhBDP and right mouse with ‘‘pH-activatable’’ Herceptin–DiEtNBDP. The tumor-to-heart ratio of the pH-activatable probe was 22-fold higher than that of the control probe (193.0 versus 8.7 arbitrary units) (14).

after ligand binding, this imaging strategy can afford a general and powerful method to diagnose and monitor the target tumors. Main potential application of the probes will be used as a clinical tool for the real-time detection of tumors during surgical resection. Such an agent also provides sufficient contrast for sensitive and reliable detection of tumors with a fluorescence endoscope (14). Finally, the strategy could be used in photodynamic therapy to salvage normal tissues and specifically enhance the cytotoxic effect on tumors.

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4. Notes 1. All SE stock solutions should be stored at –20C in the dark. Avoid moisture exposure as much as possible to keep SEs reactive. 2. Buffers (pH 8–9) containing no free amine, such as sodium phosphate buffer, are recommended. Tris and glycine that have free amines must be avoided because those molecules can react with SE. For efficient labeling, moderately basic conditions are required for aliphatic amines to become sufficiently nucleophilic by deprotonation and react with SE. Under such conditions, SE is hydrolyzed in competition with labeling; but this side reaction is usually slow below pH 9. Buffers can be prepared at any concentration as long as pH of reaction solutions is retained. 3. Do not freeze the solutions to avoid denaturation of the conjugates. The prepared conjugates are recommended to be used in imaging experiments within several days as immediately as possible. 4. Unify the condition for tumor cell preparation as possible, because the activity of tumor cells can severely influence the growth rate and pattern of tumors formed in mouse models. It is noteworthy that sub-confluent cells empirically have high viability, providing stable, satisfactory tumor models. 5. Tumor mouse models should be carefully monitored every day to establish a stable procedure for efficient models. Described number of days for breeding after tumor cell injection is just a reference, because the degree of tumor dissemination depends on the growth environment of mice, etc. Avoid overprogression of tumor model that induces suffering and eventually kills treated mice. 6. Unless trypsinized tumor cells are separated to single cells, injected mouse models will die owing to infarction of aggregated cells in capillary vessels. References 1. Hengerer, A., Wunder, A., Wagenaar, D. J., Vija, A. H., Shah, M., and Grimm, J. (2005) From genomics to clinical molecular imaging. Proceedings of the IEEE 93, 819–828. 2. Krohn, K. A., O’Sullivan, F., Crowley, J., Eary, J. F., Linden, H. M., Link, J. M., Mankoff, D. A., Muzi, M., Rajendran, J. G., Spence, A. M., and Swanson, K. R. (2007) Challenges in clinical studies with

multiple imaging probes. Nucl Med Biol 34, 879–885. 3. Becker, A., Hessenius, C., Licha, K., Ebert, B., Sukowski, U., Semmler, W., Wiedenmann, B., and Grotzinger, C. (2001) Receptor-targeted optical imaging of tumors with near-infrared fluorescent ligands. Nat Biotechnol 19, 327–331.

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Asanuma et al. 4. Moon, W. K., Lin, Y., O’Loughlin, T., Tang, Y., Kim, D. -E., Weissleder, R., and Tung, C. -H. (2003) Enhanced tumor detection using a folate receptor-targeted near-infrared fluorochrome conjugate. Bioconjug Chem 14, 539–545. 5. Weissleder, R., Tung, C. -H., Mahmood, U., and Bogdanov, A. (1999) In vivo imaging of tumors with protease-activated nearinfrared fluorescent probes. Nat Biotechnol 17, 375–378. 6. Bremer, C., Tung, C. -H., and Weissleder, R. (2001) In vivo molecular target assessment of matrix metalloproteinase inhibition. Nat Med 7, 743–748. 7. Hama, Y., Urano, Y., Koyama, Y., Kamiya, M., Bernardo, M., Paik, R. S., Shin, I. S., Paik, C. H., Choyke, P. L., and Kobayashi, H. (2007) A target cell-specific activatable fluorescence probe for in vivo molecular imaging of cancer based on a self-quenched avidin-rhodamine conjugate. Cancer Res 67, 2791–2799. 8. Kamiya, M., Kobayashi, H., Hama, Y., Koyama, Y., Bernardo, M., Nagano, T., Choyke, P. L., and Urano, Y. (2007) An enzymatically activated fluorescence probe for targeted tumor imaging. J Am Chem Soc 129, 3918–3929. 9. Hama, Y., Urano, Y., Koyama, Y., Choyke, P. L., and Kobayashi, H. (2006) Targeted optical imaging of cancer cells using lectin-

10.

11.

12.

13.

14.

binding BODIPY conjugated avidin. Biochem Biophys Res Commun 348, 807–813. Austin, C. D., De Maziere, A. M., Pisacane, P. I., van Dijk, S. M., Eigenbrot, C., Sliwkowski, M. X., Klumperman, J., and Scheller, R. H. (2004) Endocytosis and sorting of ErbB2 and the site of action of cancer therapeutics trastuzumab and geldanamycin. Mol Biol Cell 15, 5268–5282. Hongyan Li, Z. M. Q. (2002) Transferrin/ transferrin receptor-mediated drug delivery. Med Res Rev 22, 225–250. Konan, Y. N., Gurny, R., and Allemann, E. (2002) State of the art in the delivery of photosensitizers for photodynamic therapy. J Photochem Photobiol 66, 89–106. Atobe, K., Ishida, T., Ishida, E., Hashimoto, K., Kobayashi, H., Yasuda, J., Aoki, T., Obata, K. -I., Kikuchi, H., Akita, H., Asai, T., Harashima, H., Oku, N., and Kiwada, H. (2007) In vitro efficacy of a sterically stabilized immunoliposomes targeted to membrane type 1 matrix metalloproteinase (MT1-MMP). Biol Pharm Bull 30, 972–978. Urano, Y., Asanuma, D., Hama, Y., Koyama, Y., Barrett, T., Kamiya, M., Nagano, T., Watanabe, T., Hasegawa, A., Choyke, P. L., and Kobayashi, H. (2009). Selective molecular imaging of viable cancer cells with pH-activatable fluorescence probes. Nat Med 15(1): 104–109.

Chapter 6 Imaging Vasculature and Lymphatic Flow in Mice Using Quantum Dots Byron Ballou, Lauren A. Ernst, Susan Andreko, James A. J. Fitzpatrick, B. Christoffer Lagerholm, Alan S. Waggoner, and Marcel P. Bruchez Abstract Quantum dots are ideal probes for fluorescent imaging of vascular and lymphatic tissues. On injection into appropriate sites, red- and near-infrared-emitting quantum dots provide excellent definition of vasculature, lymphoid organs, and lymph nodes draining both normal tissues and tumors. We detail methods for use with commercially available quantum dots and discuss common difficulties. Key words: Quantum dots, in vivo, animals, vasculature, circulation, lymph nodes, sentinel lymph nodes, lymphatic vessels.

1. Introduction Quantum dots were first used for labeling biological specimens in 1998 (1, 2), and have been used extensively since, because of the significant advantages they hold over other types of fluorophores. They combine exceptionally high brightness, due to high extinction coefficients (>6  106 M–1cm–1 at 450 nm) and large quantum yields (routinely as high as 60% for 655 nm emitting quantum dots), narrow emission bandwidths ( 6 mg). The BRET, total luminescence, and total fluorescence were measured 60–72 h after transfection. BRET levels are plotted as a function of the total fluorescence signal (fold over background)/total luminescence signal (fold over background). The results were expressed as the mean – S.E. of seven independent experiments performed in duplicate. The curves were fit using non-linear regression or linear regression (GraphPad Prism).

5. Data should be analyzed by relative fluorescence unit (RFU) or by net BiFC, where the RFU of the negative control can be subtracted from the RFU of the samples. As can be seen in the inset to Fig. 18.3, fluorescence is only reconstituted when two proteins tagged with the complementary halves of YFP were co-expressed. 3.4. BiFC/BRET

1. cDNAs prepared for the transfection should be a mix of the protein A tagged with Renilla luciferase and protein B and C tagged with YFP (1–158) and the YFP(159–238). Initially, 0.5 mg of the RLuc construct and 1 mg of each split YFP construct should be used for transfection and the quantity optimized according to fluorescence and luminescence intensities obtained. Negative and positive controls for the BiFC/BRET experiment should also be transfected (see Note 15). Follow Steps 3.1.1–3.1.10 for transfection and preparation of cells. 2. Coelenterazine H should be diluted 1:500 in PBS+ and kept away from light, at room temperature. This solution should be discarded at the end of the experiment. 3. See Steps 3.3.2–3.3.5 for the preparation the samples and BiFC measurement. 4. Ten microliters of diluted coelenterazine H is added into each well and mixed by shaking gently the plates.

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5. Monitor BRET using the Packard Fusion Instrument, reading each well for 1 s at a PMT voltage of 100, a gain of 1, with the emission filter 450/58 nm (RLuc) and 480/LP nm (BiFC) (see Notes 16 and 17 for more details). 6. Data should be analyzed by calculating the BRET ratio, given by the light emitted at 535/25 filter relative to that passed by the 450/58 nm filter. It is also possible to express the BiFC/BRET ratio as net BiFC/BRET, subtracting the ratio of the negative BiFC/BRET control from the experimental ratio. A typical experiment is shown in Fig. 18.3. BRET was only detected when the three relevant proteins, two tagged with complementary halves, were co-expressed with a third interacting protein tagged with Rluc.

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3.5. SNAP/BRET

Although SNAP-tagged proteins have been used for FRET, this is the first report that the SNAP tag can also be used in BRET applications. Coelenterazine H oxidation leads to an emission maximum at 475 nm, thus the BG-505 substrate should be compatible for use as a BRET1 acceptor. DeepBlue C (or coelenterazine 400a) has an emission maximum of 400 nm, thus the BG-430 substrate should be compatible with BRET2 experiments. We found that BG-505 was not an efficient BRET acceptor (Fig. 18.4) while BG-430 worked in this application (Fig. 18.5). 1. Prepare cDNA mixture composed of the appropriate SNAPand Rluc-tagged constructs (normally 2 mg of each DNA is sufficient) (see Note 19). Negative and positive controls for the SNAP/BRET experiment should also be transfected (see Note 15 and Note 20). Follow Steps 3.1.1–3.1.8 for transfection. 2. Prepare BG-430 or BG-505 labeling solutions (see Note 21) at a concentration of 5 mM as follows: dilute BG stock solution (1 mM) 1:200 into a DMEM supplemented with 10% FBS (600 mL of labeling solution is required per well of a 6well plate). 3. Incubate cells with 600 ml SNAP substrate solution at 37C in 5% CO2 30 min (see Note 22). 4. Wash the cells twice with DMEM with 10% FBS (see Note 23). 5. Replace the media once more and incubate cells for another 30 min at 37C in 5% CO2 (see Note 24). 6. Replace the media one last time and incubate cells for a final 30 min incubation at 37C in 5% CO2 7. Wash cells once with DMEM with 10% FBS (see Note 25) and then wash cells twice with PBS 1  (see Note 14) 8. Resuspend cells in 90 mL of PBS 1  and load them into a 96 white optiplate. 9. Read the fluorescence of the samples using the appropriate filter set on the Fusion microplate reader (see Note 17). The BG-430 chromophore is excited at 425/20 nm (peak excitation of BG-430: 421 nm) and emission is read at 480/LP nm (peak emission of BG-430: 444 and 484 nm). The BG-505 chromophore is excited at 485/10 nm (peak excitation of BG-505:504 nm) and emission is read at 535/25 nm (peak emission of BG-505:532 nm). 10. Experiments with the BG-430 were conducted using BRET2. Coelenterazine 400 A substrate was used at a final concentration of 5 mM. Dilute the stock solution (1 mM) 1:20 into PBS 1  . Do not prepare more diluted substrate needed for more

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Fig. 18.5. BRET assays using 5 mM of BG-430 substrate. These assays were performed in HEK 293 cells 48 h post-transfection. Cells were transfected with 2 mg of HA-b2AR-SNAP and 2 mg of b2AR-RLuc cDNA and were incubated with 5 mM BG-430 substrate at for 30 min. Labeling experiments were performed based on SNAP-cell 430 protocol from Covalys, but an additional 15–30 min of washing was performed after the 30 min indicated therein. Cells were resuspended in PBS, and 5 mM BRET2 substrate coelenterazine 400 A was added to each sample before reading. Readings were performed using the Fusion microplate reader (PerkinElmer) at 410 nm for the luciferase emission and at 480 nm for the BG-430 (peak emission of BG-430: 444 and 484 nm). The BRET ratio represents the BG-430 emission counts over the luciferase emission counts. Inset: Labeling of the HA-b2AR-SNAP protein with 5 mM BG-430 substrate. Cells were resuspended in PBS and were read in a Fusion microplate reader (PerkinElmer). BG-430 chromophore was excited at 425 nm and emission was read at 480 nm (peak emission of BG-430:444 and 484 nm).

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than 2 samples at a time (see Note 26). Experiments with BG505 were conducted using BRET1. Coelenterazine H substrate was used at a final concentration of 0.2 ng/mL. The stock solution (1 mg/mL) was diluted 1:500 in 1  PBS. 11. Add 10 mL of your diluted substrate to a maximum of two samples for coelenterazine 400 A (with BG-505) and read the plate immediately. 12. For BG-430, collect signals using a 410/80 nm band pass filter for the luciferase emission and a 480/LP nm band pass filter for the BG-430 emission (peak emission of BG-505: 444 and 484 nm). For BG-505, collect signals using 450/ 58 band pass filter for luciferase emission and a 535/25 band pass filter the BG-505 emission (peak emission of the BG505: 532 nm). 13. BRET2 signals are determined by the ratio of the light emitted by the 410/80 (luciferase) over the 480/LP (BG430) band pass filters or the ratio of the light emitted by the 450/58 (luciferase) over the 535/25 (BG-505) band pass filters (see Note 27).

4. Notes 1. Except if otherwise specified, solutions should be prepared in water that has a resistance of 18.2 M -cm. 2. Ten microliters of water should be heated to 80C and 20 mg of PEI dissolved in it. The solution is cooled on ice to room temperature and the pH adjusted to pH 7 with 0.5 M HCl. The volume is adjusted to 20 mL, filtered under a biological hood, aliquoted and stored at –80C. Cell toxicity of the solution is proportional to the time that the solution is kept at room temperature, so the preparation of PEI should be as rapid as possible. 3. For best results, the humanized version of the Renilla luciferase vector should be used. This vector can be purchased from Perkin-Elmer Life and Analytical Sciences. 4. You can also use any machine that is able to read both luminescence and fluorescence emissions simultaneously. 5. Coelenterazine compounds are provided by suppliers as a yellow-orange powder that needs to be reconstituted in pure ethanol. Surprisingly, we obtain better results if the compounds are dissolved at least 1 day prior making aliquots. We recommend dissolving these compounds in ethanol by vortexing, keeping them at –20C in the original tubes for at least one day, vortexing again, and then aliquoting them.

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These products are light-sensitive so they should be kept away from light. Coelenterazine 400 A supplied by Biotium is the generic version of Deep Blue C supplied by Perkin-Elmer Life and Analytical Sciences. Both can be used for the BRET2 and give similar results. 6. Dissolve a 50-nmol vial of BG-430 in 50 mL of DMSO. Mix 10 min until BG-430 is completely dissolved (remembering to protect it from light). Store this stock solution at –20C protected from light, and it should be stable for at least 6 months under these conditions. Prepare a working solution at 5 mM in DMEM supplemented with 10% FBS. Do not prepare more working solution than will be consumed within 1 h and protect it from light. 7. Cells can be transfected when they are between 40% and 60% confluent. Below this percentage, not enough cells will survive transfection. 8. DMEM can be complemented by either 5% or 10% FBS at your discretion. The cells will grow faster in 10% FBS than in 5% FBS. 9. The total amount of DNA transfected should be the same in each sample, so empty vector can be used to maintain the DNA/well quantity constant. Do not transfect more than 10 mg of DNA per well, above that the concentration of PEI will be toxic. 10. FBS interferes with the efficiency of transfection. For this reason, cDNAs should be diluted in serum-free DMEM. Tests performed in our laboratory have shown that, over 2.5% FBS, the transfection efficiency decreased significantly, and below 2.5% FBS, cells survival is dramatically reduced. 11. Do not add the PEI/DNA solution directly to cells, as this will kill the cells. Place pipette on the side of the well and push out the solution slowly into the media surrounding the cells. Shake wells gently to be sure that the PEI/DNA solution is well mixed with the media. 12. If using a large amount of DNA, and hence a large amount of PEI, we recommend only 6-h incubations to increase survival rate. 13. We have found that warming up the plate reader by turning it on an hour prior to utilization yields more stable BRET ratios during repetitive reading of the same plate. 14. Wash cells with PBS to remove all the indicator, which quenches BRET signals. 15. As a negative control, one of the proteins of interest should be changed for another protein that is known to not interact with the other putative partner. However, this protein should be

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localized to the same subcellular compartment and expressed at the same level as the protein it replaces. For BiFC/BRET, it is useful to perform negative controls for both the BiFC and the BiFC/BRET. The positive control can be any two proteins known to interact together and it is used to confirm that the experimental system is functional. In our case, we like to use homodimerization of b2-adrenergic receptors as this interaction has been well described in the literature using BRET technology (29, 30). 16. Since BRET is a ratio of the fluorescence over luminescence, protein quantification is usually not required, because small changes in cell quantity will not affect the BRET ratios. However, the first step of the experiment is to monitor the total fluorescence and luminescence of the cells to be sure that each sample expresses approximately similar amounts of Rluc- and GFP-tagged constructs. If this is not the case, you should refer to Step 3.3.2 and quantify the proteins in each sample or change the transfection conditions if different GFP-tagged proteins are expressed. Three readings of the same plate (with the samples in duplicate) should usually be taken to determine consistency. 17. To stay within the detection range of the Packard Fusion Instrument, the RLuc counts obtained for BRET2 should be between 50,000 and 130,000 and between 20,000 and 100,000 for BRET1. If the counts are too low, it is possible to concentrate the cells in PBS+ and add more cells/volume of 90 ml. If the counts are too high, it is possible to wait several seconds before reading the plate again or to decrease the number of cells by dilution with PBS+ (always using 90 ml of PBS+/well). However, in both cases, these conditions are relevant for the Packard Fusion Instrument. If another plate reader is used, the correct conditions should be determined empirically. The maximal counts should be the highest relative luminescence units (RLU) that do not saturate the machine and the minimal counts should be the lowest RLU at which the BRET ratio remains stable. At a certain value, the ratio will increase as the RLU will decrease. In BRET2, the RLuc counts decrease rapidly; therefore, it is better to read only three or four samples at one time. 18. For BiFC experiments, the fluorescence signal is highly related to the quantity of cells. To avoid artifacts caused by differences in material quantity, the same amount of proteins or cells should be added to each well. Instead of protein quantification, it is also possible to count the cells and add the same amount for each sample.

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19. To avoid possible variation in the BRET signals resulting from fluctuations in the relative expression levels of the energy donor and acceptor, we designed transfection conditions to maintain constant BG-430 SNAP/RLuc expression ratios in each experimental set. 20. Negative controls for the SNAP/BRET assay are also essential. For an interpretable SNAP/BRET assay, untagged (SNAP) versions of proteins of interest should be labeled with substrate when co-expressed with the Rluctagged partner to confirm that BRET signals are not a result of nonspecific labeling with BG substrate or expression of the untagged protein control. Another negative control should be the SNAP-tagged partner transfected with the appropriate Rluc-tagged partner but not labeled with BG substrate to confirm that the SNAP tag itself does not influence luciferase emissions. 21. BG substrates are light sensitive. Do not prepare more diluted substrate than will be used within 1 h. The BG430 substrate concentration can vary between 2 and 20 mM. Often an increase in the substrate concentration will lead to an increase in the background and does not necessarily increase the signal to noise ratio. FBS reduces the non-specific binding of the substrate to surfaces. Labeling untransfected cells could be performed as a negative control. 22. The incubation time can vary between 15 and 60 min depending on experimental conditions, expression levels of the SNAP-tag fusion protein, and substrate concentration. Covalys recommends a 30-min incubation time. 23. If cells detach, washing steps can be performed in 1.5-mL microtubes. Detach cells and wash them up and down (gently) with DMEM with 10% FBS. Spin 3 min at 800g to remove media between each washing step and incubate microtubes at 37C when necessary. 24. Incubation times can be varied from 30 to 60 min. Longer incubation times in the same DMEM solution will not decrease the background as much as replacement of the washing solution. 25. Wash cells one last time to completely remove non-reacted SNAP substrate that has leaked out of the cells. 26. Once the substrate is added to the samples, it is oxidized rapidly. Too many samples should not be read at once, as luminescence counts will decrease rapidly.

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27. BRET ratios may vary slightly between experiments but should be comparable. At least three independent experiments should be performed before pursuing BRET saturation and competition assays to confirm the specificity of each interaction.

Acknowledgments This work was supported by grants from the Canadian Institutes of Health Research (MOP-99567) and Heart and Stroke Foundation of Quebec to T.E.H. T.E.H. is a Chercheur National of the Fonds de Recherche en Sante´ du Que´bec (FRSQ). M.R. holds a doctoral scholarship from the FRSQ. We thank Vic Rebois (NIH) for helpful discussions and we also thank Covalys for providing constructs and reagents for the SNAP tag.

References 1. Vassilatis, D. K., Hohmann, J. G., Zeng, H., Li, F., Ranchalis, J. E., Mortrud, M. T., Brown, A., Rodriguez, S. S., Weller, J. R., Wright, A. C., Bergmann, J. E., and Gaitanaris, G. A. (2003) The G protein-coupled receptor repertoires of human and mouse. Proc Natl Acad Sci USA 100, 4903–4908. 2. Cabrera-Vera, T. M., Vanhauwe, J., Thomas, T. O., Medkova, M., Preininger, A., Mazzoni, M. R., and Hamm, H. E. (2003) Insights into G protein structure, function, and regulation. Endocr Rev 24, 765–781. 3. Marinissen, M. J., and Gutkind, J. S. (2001) G-protein-coupled receptors and signaling networks: emerging paradigms. Trends Pharmacol Sci 22, 368–376. 4. Rebois, R. V., Allen, B. G., and Hebert, T. E. (2003) The targetable G protein proteome: where is the next generation of drug targets? Drug Discovery Today: Targets 3, 104–111. 5. Dupre, D. J., and Hebert, T. E. (2006) Biosynthesis and trafficking of seven transmembrane receptor signalling complexes. Cell Signal 18, 1549–1559. 6. Gingras, A. C., Gstaiger, M., Raught, B., and Aebersold, R. (2007) Analysis of protein complexes using mass spectrometry. Nat Rev Mol Cell Biol 8, 645–654. 7. Hebert, T. E., Gales, C., and Rebois, R. V. (2006) Detecting and imaging protein-protein interactions during G protein-mediated signal transduction in vivo and in situ by

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14. MacDonald, M. L., Lamerdin, J., Owens, S., Keon, B. H., Bilter, G. K., Shang, Z., Huang, Z., Yu, H., Dias, J., Minami, T., Michnick, S. W., and Westwick, J. K. (2006) Identifying off-target effects and hidden phenotypes of drugs in human cells. Nat Chem Biol 2, 329–337. 15. Mervine, S. M., Yost, E. A., Sabo, J. L., Hynes, T. R., and Berlot, C. H. (2006) Analysis of G protein betagamma dimer formation in live cells using multicolor bimolecular fluorescence complementation demonstrates preferences of beta1 for particular gamma subunits. Mol Pharmacol 70, 194–205. 16. Dupre, D. J., Robitaille, M., Richer, M., Ethier, N., Mamarbachi, A. M., and Hebert, T. E. (2007) Dopamine receptor-interacting protein 78 acts as a molecular chaperone for Ggamma subunits before assembly with Gbeta. J Biol Chem 282, 13703–13715. 17. Rebois, R. V., Robitaille, M., Gales, C., Dupre, D. J., Baragli, A., Trieu, P., Ethier, N., Bouvier, M., and Hebert, T. E. (2006) Heterotrimeric G proteins form stable complexes with adenylyl cyclase and Kir3.1 channels in living cells. J Cell Sci 119, 2807–2818. 18. Gales, C., Van Durm, J. J., Schaak, S., Pontier, S., Percherancier, Y., Audet, M., Paris, H., and Bouvier, M. (2006) Probing the activation-promoted structural rearrangements in preassembled receptor-G protein complexes. Nat Struct Mol Biol 13, 778–786. 19. Heroux, M., Breton, B., Hogue, M., and Bouvier, M. (2007) Assembly and signaling of CRLR and RAMP1 complexes assessed by BRET. Biochemistry 46, 7022–7033. 20. Gronemeyer, T., Godin, G., and Johnsson, K. (2005) Adding value to fusion proteins through covalent labelling. Curr Opin Biotechnol 16, 453–458. 21. Keppler, A., Gendreizig, S., Gronemeyer, T., Pick, H., Vogel, H., and Johnsson, K. (2003) A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat Biotechnol 21, 86–89.

22. Krayl, M., Guiard, B., Paal, K., and Voos, W. (2006) Fluorescence-mediated analysis of mitochondrial preprotein import in vitro. Anal Biochem 355, 81–89. 23. Mottram, L. F., Maddox, E., Schwab, M., Beaufils, F., and Peterson, B. R. (2007) A concise synthesis of the Pennsylvania Green fluorophore and labeling of intracellular targets with O6-benzylguanine derivatives. Org Lett 9, 3741–3744. 24. Pick, H., Jankevics, H., and Vogel, H. (2007) Distribution plasticity of the human estrogen receptor alpha in live cells: distinct imaging of consecutively expressed receptors. J Mol Biol 374, 1213–1223. 25. Tirat, A., Freuler, F., Stettler, T., Mayr, L. M., and Leder, L. (2006) Evaluation of two novel tag-based labelling technologies for site-specific modification of proteins. Int J Biol Macromol 39, 66–76. 26. Gales, C., Rebois, R. V., Hogue, M., Trieu, P., Breit, A., Hebert, T. E., and Bouvier, M. (2005) Real-time monitoring of receptor and G-protein interactions in living cells. Nat Methods 2, 177–184. 27. Michnick, S. W., Ear, P. H., Manderson, E. N., Remy, I., and Stefan, E. (2007) Universal strategies in research and drug discovery based on protein-fragment complementation assays. Nat Rev Drug Discov 6, 569–582. 28. Remy, I., and Michnick, S. W. (2007) Application of protein-fragment complementation assays in cell biology. Biotechniques 42, 137, 139, 141 passim. 29. Angers, S., Salahpour, A., Joly, E., Hilairet, S., Chelsky, D., Dennis, M., and Bouvier, M. (2000) Detection of beta 2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET). Proc Natl Acad Sci USA 97, 3684–3689. 30. Salahpour, A., Angers, S., Mercier, J. F., Lagace, M., Marullo, S., and Bouvier, M. (2004) Homodimerization of the beta2adrenergic receptor as a prerequisite for cell surface targeting. J Biol Chem 279, 33390–33397.

Chapter 19 PIN-G Reporter for Imaging and Defining Trafficking Signals in Membrane Proteins Lynn Mckeown, Vicky C. Jones, and Owen T. Jones Abstract The identification of motifs that control the intracellular trafficking of proteins is a fundamental objective of cell biology. Once identified, such regions should, in principle, be both necessary and sufficient to direct any randomly distributed protein, acting as a reporter, to the subcellular compartment in question. However, most reporter proteins have limited versatility owing to their endogenous expression and limited modes of detection – especially in live cells. To surmount such limitations, we engineered a plasmid – pING – encoding an entirely artificial, type I transmembrane reporter protein (PIN-G), containing HA, cMyc and GFP epitope, and fluorescence tags. Although originally designed for trafficking studies, pIN technology is a powerful tool applicable to almost every area of biology. Here we describe the methodologies used routinely in analyzing pIN constructs and some of their derivatives. Key words: Green fluorescent protein (GFP), live imaging, trafficking, epitope tags, reporter constructs, photoactivation, lentivirus.

1. Introduction 1.1. pIN Technology

A fundamental problem in cell biology is how best to test the contribution of putative protein interaction domains in phenomena such as trafficking, docking, and regulation, especially when they are housed within highly complex, often oligomeric, membrane or cytoplasmic parent protein structures (1). Do such domains act only in the context of the parent protein? Do they operate independently or hierarchically? Is their function regulated and if so how? Conventionally, a powerful way to address these questions has been to test whether the domain’s putative function can be transplanted onto an unrelated ‘‘reporter’’ protein (2, 3). However, available reporter proteins have serious limitations,

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including their endogenous expression, limited modes of detection, and poor experimental versatility (1–3). To circumvent such problems, we introduced a strategy – termed pIN technology – based upon pIN-G (Genbank: AY841887), a highly engineered expression plasmid, encoding an entirely artificial modular type I membrane protein (PIN-G) designed to maximize functionality in imaging and biochemical experiments (1). The design and construction of PIN-G and its encoding plasmid – pIN-G – have already been described (1), and its salient features are shown in Fig. 19.1A, B. Briefly, the pIN-G vector encodes for a fusion protein containing in order: an efficient leader sequence for membrane insertion, an external hemagglutinin (HA) epitope tag, enhanced green fluorescent protein (eGFP), a second, cMyc, epitope tag, a transmembrane spanning domain from platelet-derived growth factor receptor (PDGFR), and a short carboxy terminus. Together, these elements afford a 30-kDa integral protein with a type I transmembrane topology. In addition, the pIN-G vector contains two, non-overlapping, multiple cloning sites (MCS-1 and MCS-2) located in sequences encoding non-functional regions of the extracellular and intracellular protein domains. Consequently, MCS-1 allows the facile introduction of sequences encoding motifs which can interact with components that are extracellular or in the lumen of the secretory pathway (4). Conversely, MCS-2 permits the introduction of sequences encoding motifs that have a cytoplasmic disposition (1).

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Fig. 19.1. Features of PIN-G, and some of its derivatives. (A) Cartoon showing type I transmembrane (extracellular amino terminus, single pass) topology and modular features of PIN-G following signal peptide cleavage. Salient features include the HA and cMyc epitope tags, green fluorescent protein (GFP) fluorophore, and the transmembrane domain from the platelet-derived growth factor receptor (PDGFR). (B) Schematic showing main features of the pIN-G expression plasmid, including (small boxes) the CMV promoter (CMV), kozak (KZ) consensus sequence, multiple cloning sites (M1 and M2), and polyadenylation sequences. Protein-encoding regions are shown in large boxes (notation as in A plus the Ig chain signal peptide (SP)). (C) Protein-encoding elements of two PIN-G derivatives – PIN-ANT – which is targeted to the anterograde secretory pathway and PIN-KDEL – a derivative retained in the lumen of the endoplasmic reticulum by virtue of a C-terminal KDEL sequence (4). All notation as in B.

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Owing to its modular design and unique cloning sites, one may introduce, substitute, or remove select protein encoding regions into the extracellular, transmembrane, or intracellular domains and test their function. Daughter pIN-G derivatives can be generated with new properties permitting their application as parents for further derivatives. Both approaches are exemplified in the twostage construction of pIN-KDEL, a reporter designed to image endoplasmic reticulum dynamics (4) (Fig. 19.1C). Here, a premature stop codon was first engineered into the parent pIN-G vector to yield a truncated protein lacking the cMyc tag and the entire transmembrane and N-terminal sequences of pIN-G. Owing to retention of the amino terminal signal peptide, the resulting protein – termed PIN-ANT – translocates into the ER and, following signal peptide cleavage, is released as a soluble HA and GFP-tagged protein capable of progressing through and

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Fig. 19.2. Fluorescence imaging of PIN-G, and PIN-R-Ant – a red (RFP) fluorescent derivative of PIN-G targeted to the anterograde secretory pathway in HEK293 cells. (A) Expression of total PIN-G fluorescence determined using its intrinsic GFP fluorophore. Note strong cell surface fluorescence (arrowheads) as well as some puncta within intracellular compartments. In this sample, the nuclei were counterstained using DAPI (see Note 7). (B) Example of the use of anti-HA labelling against the extracellular HA epitope tag in pIN-G to resolve just surface rather than total (surface+intracellular) PIN-G distribution (see 3.2). (C) and (D) Co-expression of pIN-G (panel C) and anterograde secretory pathway-targeted pIN-R-Ant (panel D, same cell as in C). Note fluorescence for PIN-G (panel C) as in panel A, but without nuclear (DAPI) counterstaining.

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demarcating the lumen of the anterograde secretory pathway (Fig. 19.2). Conversion of the functionally inert C-terminal residues of PIN-ANT to a KDEL (Lys-Asp-Glu-Leu-COOH) ER retention motif yields an HA and GFP-tagged protein – termed PIN-KDEL – which selectively resides and demarcates the ER lumen. Using lentiviral gene transduction technology, PINKDEL serves as a useful marker of ER dynamics and continuity in neurons (4). It is also possible to generate pIN constructs with altered intrinsic reporter properties. While obvious modifications include substitutions of the existing HA and cMyc epitope tags, the most important involve those that use sequences encoding monomeric fluorescent proteins with select spectral properties for multicolor imaging applications (5). One additional arena that holds much promise is the development of pIN constructs tailored for fluorescence photoactivation (FPA) studies of protein dynamics in cells (6). Using a photoactivatable GFP (PA-GFP) analog that differs from GFP in just four amino acids (L64F, T65S, V163A, T203H) (7), it has been possible to prepare a construct encoding a photoactivatable analog of the ER-targeted pIN-KDEL termed PA-PIN-KDEL which, following brief excitation at 413 nm, shows a 100-fold increase in fluorescence intensity when imaged with 488 nm light (Fig. 19.3). By tracking the dispersion of activated PA-PINKDEL, it has been possible to confirm the continuity of the ER in diverse cells suggested by photobleaching studies.

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Fig. 19.3. Photoactivation of ER-targeted PIN-construct containing photoactivatable GFP (PA-GFP) (A) Fluorescence pre-activation. (B) Fluorescence post-activation. In these experiments, HEK293 cells were transduced with PA-PIN-KDEL (See Fig. 19.1C) using the lentiviral gene delivery system. After 48 h, the cells were imaged at (excitation 488 nm; emission 505 nm), then irradiated for 10 s by a laser (413 nm excitation) in the region approximated by the ellipse in panel A, and then re-imaged. To preclude rapid fluorescence dissipation due to PIN-KDEL diffusion, the sample shown here was fixed prior to irradiation. For dynamic live imaging studies, the fixation step would be omitted and the sample imaged intermittently.

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In this chapter we now describe the methods we use, routinely, for analyzing the distribution and surface expressions (e.g. Fig. 19.4) of pIN constructs in mammalian cells.

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Fig. 19.4. Determination of surface versus intracellular PIN construct distribution. (A) Assay principle: Any pIN construct (pIN) expressed at the cell surface is detected by labeling live cells with primary antibodies (Po) to the extracellular HA epitope tag followed by Cy3-conjugated secondary (So) antibody. Total PIN construct expression (surface+intracellular) is determined from the GFP (G) fluorescence. (B) Fluorescence cytometry of cells showing red (Cy3) and green (GFP) fluorescence corresponding to surface and total construct expression. Surface expression is determined from the number of cells showing surface (Cy3) to total (GFP) fluorescence (quadrant R4/R4+R6). Note: Cy3 and GFP fluorescence intensities are expressed on log scales. Quadrants R3 and R5 denote background red and green fluorescence, respectively.

2. Materials 2.1. Cell Culture and Transfection

1. HEK293 cells (ECACC) propagated in T75 vented cell culture flasks. 2. Culture medium: Dulbecco’s minimum essential medium (DMEM) supplemented with 10% fetal calf serum (v/v), 2 mM L-glutamine (w/v), and 1% (w/v) penicillin and streptomycin. 3. Phosphate-buffered-saline without calcium and magnesium (PBS–). 4. 6-well culture plates. 5. Transfection medium: unsupplemented DMEM. 6. Fugene 6 transfection agent (Roche Applied Sciences, UK).

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2.2. Immunofluorescence for Imaging and FACS

1. Round 10 mm, no. 1.5 glass coverslips. 2. Collagen solution: Type I rat-tail collagen (Sigma, Gillingham, UK) 1:400 in PBS–. 3. Phosphate-buffered saline with calcium and magnesium (PBS+). 4. Paraformaldehyde fixative: 4% solution (w/v) in PBS– fresh or stored at –20C. 5. Quench solution: 0.1 M glycine in PBS–. 6. Saponin solution: 0.5% (w/v) saponin (Sigma, UK) in PBS–. 7. Primary antibodies: mouse monoclonal anti-HA.11 (Covance, Cambridge Biosciences, UK), mouse monoclonal antimyc (Developmental Studies Hybridoma Laboratories, Iowa, USA). 8. Secondary antibodies: Cy3-conjugated anti-mouse and Cy5conjugated anti-mouse for FACS (Jackson Immunoresearch, Stratech, UK). 9. Antibody dilution buffer: non-permeabilized cells: PBS–; permeabilized cells: 0.01% (w/v) saponin in PBS–. 10. Nuclear stain: 0.05 mg/mL 40 -6-diamidino-2-phenylindole (DAPI) in PBS–. 11. Mountant: ProLong Gold Fade (Molecular Probes, Invitrogen, Gillingham, UK).

2.3. FACS Analysis

1. T25 vented cell culture flasks. 2. Wash buffer: PBS+ containing 1% (v/v) fetal calf serum.

2.4. Lentiviral Transduction of pIN Constructs

1. HEK293 FT cells (Invitrogen, UK). Culture medium – see Section 2.1. 2. Phenol red-free culture medium: DMEM, containing 25 mM glucose, without phenol red supplemented with 10% (v/v) FBS, 2 mM L-glutamine, 100 U/mL penicillin and 100 mg/mL streptomycin. 3. Lentiviral packaging plasmids (8): pMDLg/pRRE (core plasmid), pRSV.REV (core plasmid), and pMD2VSV-G (envelope plasmid). Self-inactivating lentiviral transfer vector plasmid (e.g., pLV-pIN-KDEL). All the above plasmids are available from the authors upon request. 4. Calcium phosphate mammalian transfection kit (Takara Bio Europe, San-Germain-en-Laye, France). 5. 0.22-mm syringe filter. 6. Vivaspin 20 100,000 molecular weight cut-off filter tubes (Sartorius Ltd., Epsom, UK).

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3. Methods 3.1. Transfection of Cells with pIN Constructs

1. When approaching confluence (every 3–4 days), passage HEK293 cells by removing the culture medium and suspending the cell monolayer with PBS–. For maintenance cultures seed new T75 flasks at 1:20 in fresh culture medium. 2. For experimental cultures, seed at 1:50 into fresh culture medium using 6-well plates. Use 1 well/data point for pulse-chase and FACS experiments or 1 well. For imaging, assume 1 well/3 coverslips. 3. Incubate at 37C 5%CO2 until cells attain 50% confluence. 4. For each well of cells to be transfected, first mix 2 mg of pIN construct cDNA with 150 mL transfection medium in a 0.5-mL microcentrifuge tube. In a separate 0.5 mL microcentrifuge tube, add 6 mL of pre-warmed (22C) Fugene 6–150 mL transfection medium (see Note 1). 5. Combine the DNA and Fugene 6 mixtures, tap gently, and leave the transfection mixture 20 min for lipid–DNA complexes to form. 6. Apply the transfection mixture evenly and dropwise to the cell well (see Note 2). 7. Incubate cells at 37C for 24 h.

3.2. Immunostaining of Transfected Cells to Determine Total and Surface Expression of pIN Constructs

1. Using a microbiological safety cabinet, place sterile ethanolwashed coverslips in a 12-well plate and wash three times with PBS+ to remove all traces of ethanol. 2. To promote HEK293 cell adherence, add 0.5 mL collagen solution to each well/coverslip for >1 h (see Note 3). 3. Prior to use, aspirate the collagen solution and wash the coverslips twice with 0.5 mL PBS+, then add 1 mL of prewarmed (37C) cell culture medium to each well. 4. At 24-h post-transfection (see Section 3.1. Step 7), aspirate the transfection medium and detach cells in 1 mL PBS– by trituration (see Note 4). For imaging seed cells at 1:3 per coverslip, then incubate for a further 24 h at 37C. For FACS analysis use cells without splitting. 5. To detect total pIN-construct expression via its reporter (e.g., GFP in the case of pIN-G constructs) fluorescence without further staining, the cells from Step 4 can be fixed directly (Step 13). To visualize total pIN construct expression immunocytochemically rather than from the reporter fluorescence, go to Step 6. To distinguish surface from total PIN construct expression, go to Step 10.

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6. Permeabilize the cells from Step 4 by adding 1 mL/well 0.5% saponin solution for 10 min at 22C. (see Note 5). 7. Aspirate saponin solution, add 250 mL/well of anti-HA 1:500 in dilution buffer and incubate at 22C for 30 min. 8. Wash cells carefully three times with PBS+, then add 200 mL Cy3-conjugated anti-mouse secondary antibody, 1:200 in dilution buffer, for 20 min at 22C. 9. Wash three times with PBS+, then proceed to fixation Step 13. 10. To distinguish surface from total PIN construct expression (see Note 6), incubate each well of cells from Step 4 with 250 mL anti-HA (1:500 in dilution buffer) for 30 min at 22C. 11. Wash cells carefully three times with PBS+, then add 200 mL secondary Cy3-conjugated anti-mouse 1:200 in dilution buffer for 20 min at 22C. 12. Wash cells three times with PBS+, then proceed to fixation Step 13, below. 13. Aspirate the medium from the coverslips and fix cells with 0.5 mL of paraformaldehyde fixative for 20 min (see Note 7). 14. Remove fixative and wash cells three times with PBS+ (see Note 8). Inactivate any remaining fixative with 1 mL of quench solution for 10 min. Remove quench solution by washing coverslips twice with PBS+. 15. Pipette 6 mL of mountant (prewarmed to room temperature) onto a clean glass microscope slide. 16. Carefully remove the coverslip from the well with watchmaker’s forceps, using a clean tissue wipe off excess fluid from the edge of the coverslip and place it, cell side down, onto the mountant. 17. Cover and leave mounted slide, in dark, to ‘‘cure’’ for > 2 h, ideally overnight. 18. Seal the coverslip edges with clear nail varnish. 19. Image within 1 week, if possible, using any fluorescent microscope equipped with filter sets for GFP or Fluorescein isothiocyanate (FITC) (see Note 9).

3.3. Quantitation of Surface and Total pIN Construct Expression by Flow Cytometry

1. At 24-h post-transfection (see 3.1. Step 4), aspirate the transfection medium and detach cells in 1 ml PBS– by trituration (see Note 4). Seed cells into a T25 flask containing 6 mL culture medium and incubate for a further 24 h at 37C. Ensure there are enough cells for assay and control (see Step 10) samples.

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2. Place cells on ice to prevent internalization and cool to 4C (see Note 10). Once cooled, carefully decant the medium. To each T25 flask add 2 mL of anti-HA diluted 1:500 in FACS wash buffer (chilled to 4C) and incubate on ice for 45 min. 3. Detach cells (see Note 4) using a plastic pipette and transfer cell suspension into glass tubes and pellet cells by low-speed centrifugation at 4C. 4. Decant the medium and wash cell pellets by resuspending in 0.5 mL wash buffer and centrifugation. Repeat centrifugation wash step two more times. 5. Dilute Cy5-conjugated anti-mouse secondary antibody (see Note 11) 1:1000 in a polypropylene tube with wash buffer (5 mL antibody and 995 mL wash buffer is enough for 10 samples), mix and place on ice. 6. Add 100 mL diluted antibody to each FACS sample tube (Step 4). Gently resuspend cells and incubate on ice for 30 min. 7. Pellet cells by centrifugation and decant off the antibody supernatant. 8. Wash cells, as in Step 4, twice with 0.5 mL ice-cold wash buffer and 1  with PBS–. 9. Resuspend the cell pellet in 100 mL PBS–, then fix cells by adding 100 mL 2% (w/v) paraformaldehyde followed by 200 mL PBS+. 10. Perform FACS analysis (see Note 12) taking mean fluorescent values of experimental samples and controls, which should minimally include transfected cells treated, as above, but omitting the primary antibody (in Step 2). 11. Surface expression is assessed from the ratio of the number of cells showing surface (e.g., Cy5) fluorescence to the number of total number of cells expressing PIN construct fluorescence (e.g., green for pIN-G constructs) (Fig. 19.4). 3.4. Pulse-Chase Assay for Imaging pIN Construct Internalization

Prior to fixation, cells can be subjected to a pulse-chase procedure in order to visualize pIN-construct internalization. 1. Follow 3.2. Steps 1–4. 2. Place cells on ice to prevent internalization and cool to 4C. Carefully aspirate off the medium and add 250 mL /well of anti-HA antibody diluted 1:500 in PBS+ (chilled to 4C). Incubate samples on ice for 45 min. 3. Aspirate antibody solution and wash twice with PBS+. Add 1 mL of culture medium (warmed to 22C) and transfer cells to a 37C incubator to initiate internalization.

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4. After the desired internalization time period, e.g., 1 h (see Note 13), immediately, place the cells on ice, aspirate the medium from the coverslips, and fix the cells with 0.5 mL paraformaldehyde fixative for 20 min. 5. Remove the fixative by gentle aspiration and wash the cells three times with PBS. 6. Inactivate any remaining fixative with 1 mL of quench solution for 10 min, then wash cells three times with PBS+. 7. Add 200 mL of Cy3-conjugated anti-mouse secondary antibody 1:200 in dilution buffer for 20 min at 22C. 8. Wash three times with PBS+, then mount coverslips as described in Steps 15–19, Section 3.2. 3.5. Pulse-Chase FACS Assay of pIN Construct Internalization

Through a combination of flow cytometry and pulse-chase protocols, it is possible to quantify pIN construct internalization from the cell surface, accurately and conveniently. 1. Follow 3.3. Steps 1 and 2. 2. Carefully decant anti-HA antibody and wash cells twice with PBS+. 3. Add 3 ml of culture medium (22C) and incubate at 37C for desired internalization time period (see Note 13). 4. Follow procedures in Section 3.3 Steps 3–11, ensuring cells maintained at 4C

3.6. Viral Transduction of pIN Constructs

3.6.1. Lentivirus Preparation

Owing to the moderately small size of pIN reporter constructs, pIN technology is especially suited for viral gene expression, where insert packaging can be an issue. Although the constructs can be packaged into almost any viral gene delivery system, we use a fourth-generation, replication-defective lentivirus system tailored for gene therapy and transduction of cells, such as neurones, which are notoriously difficult to transfect using conventional lipid-based methods. To enhance pleiotropy, the lentivirus is pseudotyped with VSV-G coat protein, although other coats can be used (see Note 13). 1. HEK293 FT cells (a cell line specialized for viral production) are grown to 40–60% confluency in T75 flasks, exactly as described for HEK293 cells in Section 3.1, Step 1. 2. At 1–2 h prior to transfection, replace the culture medium with 20 ml fresh pre-warmed phenol red-free culture medium. 3. To package VSV-G (Indiana serotype) pseudotyped lentiviral particles, the HEK293FT cultures are transfected using a calcium phosphate mammalian transfection kit as per the

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manufacturer’s instructions. Good transfection can be achieved using the following ratios: 15 mg pMDLg/pRRE, 5.8 mg pRSV.REV, and 8.2 mg pMD2VSV-G, plus 23.4 mg of the appropriate self-inactivating lentiviral transfer vector plasmid (e.g., pLV-pIN-KDEL). 4. At 16 h post-transfection, remove the culture medium and add 12 ml fresh pre-warmed phenol red-free medium (see Note 14). 5. At 24–48 h post-transfection, collect the culture medium (1st batch). Add 8 ml of fresh, pre-warmed, phenol red-free medium to the T75 flask and return to the incubator. 6. Centrifuge the medium from Step 5 at 500g for 3 min to remove dead cells, then remove minor traces of cell debris by passing the centrifuged medium supernatant through a 0.22 mm Millex1 GP syringe filter. 7. Store the virus-containing medium supernatant from Step 6 in a sealed tube at 4C (or at –80C for longer-term storage). 8. After a further 24 h, i.e., at 48–72 h post-transfection, re-harvest the culture medium (2nd batch) from Step 5 and repeat Steps 6 and 7. 9. Pool first and second batches of spun/filtered culture medium and concentrate to a volume of 300 mL by centrifugation at 5000g, 4C, in a Vivaspin 20 100,000 molecular weight cut-off filter tube. 10. Store the concentrated virus in aliquots of 100 mL batches – 80C until required. The virus will stay active for at least 6 months. 3.6.2. Lentiviral Transduction

1. Prepare HEK293 cells for experimentation as described in Section 3.1, Step 1. Other cells may be used providing they are not >50% confluent. 2. At 24 h after plating, transduce HEK293 cells by adding 50–100 ml of thawed concentrated virus stock (3.6.1. Step 9) directly to the culture medium and swirl gently to mix. 3. Incubate cells at 37C for 24 h, then process for imaging, FACS (e.g., as in 3.2–3.5) or biochemistry.

3.7. Photoactivation Experiments with PA-pIN Constructs

1. HEK293 cells, transfected (See 3.1) or transduced (See 3.6.2) with the desired PA construct, should be prepared for live imaging by growing in glass-bottomed culture dishes coated with type I rat-tail collagen (see Notes 2 and 15). 2. Pre-activated PA-PIN-KDEL may be visualized by excitation using a low-power 413 nm laser line. Unlike WT GFP, PAGFP shows negligible fluorescence when excited at 488 nm (i.e., GFP imaging mode) (see Note 16).

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3. Photoactivate PA-GFP-tagged pIN construct using focal illumination with high-intensity 400–450 nm light for 5–30 s. Visualize activated PA-GFP by switching to 488 nm, imaging mode excitation with continuous or intermittent (e.g., every 15 s), image capture.

4. Notes 1. Undiluted Fugene 6 must not come into contact with plastic surfaces other than the pipette tip. A 3:1 Fugene 6:cDNA ratio is optimal for efficient transfection of HEK293 cells with pIN constructs. Follow the manufacturer’s instructions to optimize transfections for other cell types. 2. There is no need to change the incubation medium already present on the cells. 3. Depending on cell type, the coverslips may go uncoated or another extracellular matrix protein may be used. 4. HEK293 cells can be detached using PBS–; more adherent cells may require trypsin or EDTA (for FACS analysis) solutions. 5. Saponin is a mild permeabilization agent affording excellent preservation of lipid structures in cells. However, as its actions can be reversible, all post-permeabilization antibody and wash solutions should contain 0.01% (w/v) saponin. 6. Discrimination of surface versus total expression of PIN constructs exploits the presence of its extracellular HA (or cMyc) epitope tags and the failure of antibodies to penetrate nonpermeabilized cells. 7. To facilitate focusing, we recommend treating the cells with 0.5 mL nuclear stain solution prior to fixation. After 1 min, aspirate off the stain and add 0.5 mL PBS– to the well. 8. Carefully apply wash solutions down well sides rather than onto the cells directly. 9. We use a DeltaVision restoration workstation (Applied Precision Instruments, Seattle, USA) which utilizes a Unix-based computer system equipped with SoftWorx version 2.5. Using the high-precision nanochassis stage, optical sections of 0.2 mm are acquired through the z plane of the cells. Z stacks are generated and then deconvolved using a constrained iterative algorithm assigned by DeltaVision.

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10. To isolate the fraction of pIN construct that is at the cell surface, the cells are labeled with antibodies to the extracellular HA-tag (see Note 6). Chilling at 4C during the labeling period suppresses internalization and, thus, overestimation of surface expression. 11. The antibody-conjugated fluorophore is chosen depending on the laser lines available on the FACS cytometer, with the only requirement being good separation of its fluorescence emission from that of the pIN construct. For pIN-G constructs (green, GFP fluorescence), we use Cy3 or Cy5-conjugated antibodies routinely. 12. We use a FACScaliber flow cytometer (Becton Dickinson, UK). 13. Since the rate of internalization will depend upon the specific pIN construct, pilot time course studies should be performed. Incubation times of 10–100 min are typical. 14. All steps involving live virus (4.6., Step 4 onward) must be conducted in accordance with local guidance for viral handling. In the United Kingdom, this is currently ACDP containment level 2, i.e., requiring procedures to be conducted in a level II microbiological safety cabinet in an ACDP level 2 classified room. Although lentiviral particles are killed on exposure to air and dessication, both the user and the environment are potentially at risk from viral particles in aerosols or liquids. Precautions include wearing two pairs of gloves, of which the outer pair is changed regularly (the inner pair forms a ‘‘second skin’’), and a Howie-style laboratory coat at all times. Any liquid waste should be treated with an antiviral agent, such as VirkonTM (DuPont, Stevenage, UK), prior to disposal in waste water systems. Solid waste should be autoclaved prior to incineration. Equipment and surfaces should be disinfected with a recognized antiviral agent (e.g., VirkonTM) followed by 70% ethanol. 15. When using viral transduction, replace culture medium entirely with phenol red-free culture medium as the indicator can be phototoxic to cells. 16. To circumvent difficulties in focusing (due to low pre-activation PA-GFP fluorescence), cells can be co-transfected with a second plasmid encoding an additional, but more discernible fluorophore such as mRFP.

Acknowledgments This work was supported by funds from the Biotechnology and Biological Sciences Research Council UK: BBSRC, BB/ D008891/1. We are indebted to Professor P.-L. Nicotera and

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Dr. D. Bano (University of Leicester, UK) for their gift of the lentiviral packaging system and advice on its use. We also thank Jane Kott and the University of Manchester Bioimaging Facility for imaging support. References 1. McKeown, L., Robinson, P., Greenwood, S. M., Hu, W., Jones, O.T, (2006) PIN-G - a novel reporter for imaging and defining the effects of trafficking signals in membrane proteins. BMC Biotechnol 6, 15. 2. Bonifacino, J. S., Cosson, P., and Klausner, R. D. (1990) Colocalized transmembrane determinants for ER degradation and subunit assembly explain the intracellular fate of TCR chains. Cell 63, 503–513. 3. Gu, C., Jan, Y. N., and Jan, L. Y. (2003) A conserved domain in axonal targeting of Kv1 (Shaker) voltage-gated potassium channels. Science 301, 646–649. 4. Jones, V. C., McKeown, L., Verkhratsky, A., Jones, O. T. (2008) LV-pIN-KDEL: a novel lentiviral vector demonstrates the morphology, dynamics and continuity of the

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endoplasmic reticulum in live neurones. BMC Neurosci 9, 10. Prescott, M., Battad, J. M., Wilmann, P. G., Rossjohn, J., and Devenish, R. J. (2006) Recent advances in all-protein chromophore technology. Biotechnol Annu Rev 12, 31–66. Patterson, G. H. (2008) Photoactivation and imaging of photoactivatable fluorescent proteins. Curr Protoc Cell Biol Chapter 21: Unit 21.6. Patterson, G. H., and Lippincott-Schwartz, J. A. (2002) Photoactivatable GFP for selective photolabeling of proteins and cells. Science 297, 1873–1877. Follenzi, A., and Naldini, L. (2002) HIVbased vectors. Preparation and use. Methods Mol Med 69, 259–274.

Chapter 20 Imaging b-Galactosidase Activity In Vivo Using Sequential Reporter-Enzyme Luminescence Georges von Degenfeld, Tom S. Wehrman, and Helen M. Blau Abstract Bioluminescence using the reporter enzyme firefly luciferase (Fluc) and the substrate luciferin enables noninvasive optical imaging of living animals with extremely high sensitivity. This type of analysis enables studies of gene expression, tumor growth, and cell migration over time in live animals that were previously not possible. However, a major limitation of this system is that Fluc activity is restricted to the intracellular environment, which precludes important applications of in vivo imaging such as antibody labeling, or serum protein monitoring. In order to expand the application of bioluminescence imaging to other enzymes, we characterized a sequential reporter-enzyme luminescence (SRL) technology for the in vivo detection of b-galactosidase (b-gal) activity. The substrate is a ‘‘caged’’ D-luciferin conjugate that must first be cleaved by b-gal before it can be catalyzed by Fluc in the final, light-emitting step. Hence, luminescence is dependent on and correlates with b-gal activity. A variety of experiments were performed in order to validate the system and explore potential new applications. We were able to visualize non-invasively over time constitutive b-gal activity in engineered cells, as well as inducible tissue-specific b-gal expression in transgenic mice. Since b-gal, unlike Fluc, retains full activity outside of cells, we were able to show that antibodies conjugated to the recombinant b-gal enzyme could be used to detect and localize endogenous cells and extracellular antigens in vivo. In addition, we developed a low-affinity b-gal complementation system that enables inducible, reversible protein interactions to be monitored in real time in vivo, for example, sequential responses to agonists and antagonists of G-protein-coupled receptors (GPCRs). Thus, using SRL, the exquisite luminescent properties of Fluc can be combined with the advantages of another enzyme. Other substrates have been described that extend the scope to endogenous enzymes, such as cytochromes or caspases, potentially enabling additional unprecedented applications. Key words: b-galactosidase, luminescent imaging, in vivo pharmacology, G-protein-coupled receptor.

1. Introduction 1.1. Bioluminescence Imaging of Firefly Luciferase

Bioluminescent imaging based on firefly luciferase (Fluc) activity is now a well-established method that has proven to be a valuable tool for the investigation of biological and pharmacological

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questions (1–5). The characteristics, strengths, and limitations of the different luciferases and their respective luminescent substrates have been described and discussed in detail elsewhere (6). In principle, bioluminescence has the potential to provide unsurpassed sensitivity due to the absence of endogenous luciferase expression in mammalian cells and to the exceedingly low background luminescence emanating from animals. 1.2. Luminescence Imaging of Other Peptidases and Proteases by Sequential ReporterEnzyme Luminescence

We have developed a technology that increases the versatility of luminescence imaging by making it possible for the first time to non-invasively image the activity of enzymes other than luciferases, while capitalizing on the advantages of luciferase-based bioluminescence (7). The method is based on ‘‘caged’’ luciferin conjugates that cannot be cleaved by Fluc due to the presence of a bulky side group. Following cleavage of the side group at a cleavage site specific to the target enzyme, free D-luciferin is generated that, subsequently, is catalyzed by constitutively expressed firefly luciferase to produce light (7). Fluc no longer acts as a bona fide reporter enzyme, but rather as a secondary detection system that makes it possible to visualize the activity of the enzyme of interest. Hence, enzymes for which luminescent substrates are either not known or not applicable to live cells or animals become amenable to bioluminescent imaging. Such a technique has the potential to greatly expand the scope of bioluminescent imaging applications. The technique was first tested in a series of proof-of-principle experiments to image b-gal, a well-known reporter enzyme, using the luciferin conjugate 1-O-galactopyranosyl-luciferin (Lugal) as described below. This substrate was first described by Miska & Geiger, and applied to the highly sensitive detection of bacterial contamination of food stocks (8–10). We provided the first evidence that Lugal has the ability to penetrate living cells without causing overt toxicity (7). As a result, it can be used to image intraas well as extracellular b-gal in cell cultures as well as in living mice, as described below. Importantly, a wide variety of novel luciferin conjugates have been developed, which make it possible to detect and quantify other proteases and peptidases, including endogenous enzymes. The following list shows selected examples of such substrates: l cytochromes P450 (differentiating between specific isoforms CYP1A1, –1A2, –1B1, –2C8, –2C9, –2C19, –2D6, –2J2, –3A4, –3A7, –4A11, –4F3B, –4F12, and –19) (11) (available from Promega, Madison, WI) l

caspases 3/7, 8, and 9 (12)

l

alpha-chymotrypsin (13)

l

carboxylic esterase (10)

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arylsulfatase (10)

l

alkaline phosphatase (10)

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kinases, e.g., carboxypeptidases A, B, and N (10, 14) Based on the principle exemplified by b-gal, it should be possible to generate substrates containing suitable peptide sequences that make it possible to detect the activity of a wide variety of peptidases or proteases via bioluminescence imaging, enabling novel applications in the fields of toxicology and pharmacology as well as to study of organ and tissue physiology and pathology. To date, however, only b-gal has been tested and shown to be amenable to bioluminescent imaging of living cells and live animals. Recently, caspase activity was imaged in living mice that, however, died later in the process of imaging, apparently due to substrate toxicity (12). Different substrate conjugates may differ from Lugal with respect to toxicity, plasma stability, ability to penetrate the membrane of intact cells, and with respect to their pharmacokinetics following intraperitoneal (or intravenous) injection. Consequently, each substrate will need to be rigorously characterized and optimized in order to assess its applicability to in vivo imaging. For b-gal, we have conducted a series of proofof-principle experiments that demonstrate the suitability of the Lugal substrate for in vivo imaging of intra- and extracellular b-gal activity repeatedly over time. l

1.3. Characterization and Applications of b-Galactosidase Bioluminescent Imaging

b-gal is one of the most widely used reporter enzymes in life sciences. The bacterial enzyme, encoded by the LacZ gene, possesses remarkable stability, retaining high activity through tissue fixation protocols and harsh chemical treatments, making it in many ways an ideal reporter system. It can be used as a reporter in cells and in transgenic animals or as a protein that can be linked to a wide variety of chemical and biological molecules. We have performed the following experiments to establish the feasibility and validity of using b-gal for in vivo bioluminescent imaging: Differentiation of LacZ expressing cells by luminescence in vitro – Lugal was applied to living cells expressing Fluc alone (Fluc cells) or LacZ and Fluc (LacZ-Fluc cells): LacZ cells incubated with Lugal were shown to produce a luminescent signal that was specific for LacZ-Fluc cells (i.e., not detectable in Fluc cells) which was linear with increasing cell number (7). Differentiation of LacZ expressing cells by luminescence in vivo – LacZ-Fluc and Fluc cells were implanted into the muscle or subcutaneously in nude mice and imaged by intraperitoneal injection of Lugal and bioluminescent imaging. LacZ-Fluc cells were shown to produce robust luminescence with a high signal-to-noise ratio compared to Fluc cells (Fig. 20.1).

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Fig. 20.1. Luminescent imaging of b-gal activity in living mice using Lugal. b-gal expressing cells can be imaged in a living subject using Lugal. One million cells expressing Fluc were injected into the left tibialis anterior (TA) leg muscle of a BALB/c nude mouse; the same number cells expressing Fluc and b-gal were injected into the right TA. Lugal was injected 6 h later, showing a clear luminescent signal over the right leg injected with cells expressing b-gal and FLuc, whereas the left leg implanted with cells expressing Fluc, but not b-gal, showed only minimal luminescence (left panel). Luciferin was injected 24 h later, showing that equivalent cell numbers expressing Fluc were present in both legs (right panel). The results are representative of five independent experiments. Bioluminescent images are quantified in photons/sec/cm2. Reproduced with permission from Nature Methods, vol. 3, no. 4, pp. 295–301 (2006).

Imaging of inducible LacZ expression in transgenic mice in vivo – Myf-5-LacZ mice were crossed with mice expressing Fluc in all cells, muscle damage was induced by notexin and imaging was performed repeatedly over a period of 9 days by intraperitoneal injection of Lugal. Inducible tissue-specific, gene expression was clearly detected. Detection of antibodies to extracellular or membrane proteins in living mice – Antibodies to a membrane protein, CD4, coupled to b-gal revealed the lymph nodes and spleen (Fig. 20.2). The activity of b-gal outside the cells cleaved the Lugal substrate releasing luciferin that entered neighboring cells, serving as a luminescent substrate for intracellular luciferase. Detection of protein-protein interactions in live animals through imaging of -gal complementation – Cells engineered to express a GPCR fused to a small fragment and b-arrestin2 fused to

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Fig. 20.2. Luminescent imaging extracellularly labeled cells and of lymphocyte distribution in vivo using anti-CD4 antibodies labeled with b-gal. (A) Injected cells extracellularly labeled with the b-gal reporter enzyme can be imaged using SRL. Untransduced C2C12 myoblasts, not expressing FLuc, were labeled with biotin, followed by an avidin-b-gal conjugate, and injected into a transgenic mouse constitutively expressing Fluc. Lugal was injected 24 h later, resulting in a robust signal localized to the site of implantation. (B–E) Detection of endogenous CD4+ T-cells by injection of anti-CD4 antibodies conjugated to b-gal in wild-type mice transplanted with the bone marrow from transgenic mice constitutively expressing Fluc. (C) Firstly, the pattern of FLuc activity from the FLuc-bone marrow transplanted mice was determined by Luciferin injection. Luminescence intensity and distribution were similar in both mice and revealed only minimal enhancement over organs containing high densities of blood-derived cells, e.g., the liver (‘‘blood pool’’). (D) The following day the same mice were injected with an anti-CD4 antibody conjugated to b-gal or a control anti-rat antibody similarly labeled with b-gal. Four hours after antibody injection, Lugal was injected intraperitoneally, revealing markedly different antibody distributions in both mice. A clear luminescent signal emerged over the cervical lymph nodes and the spleen of the mouse injected with the CD4 antibody (arrows, right panel), whereas only weak regional luminescence was seen in the mouse having received the control antibody (left panel). Bioluminescent images are quantified in photons/sec/cm2. (E) Quantification of luminescence after Lugal injection in regions of CD4+ T-cell enrichment. No difference was observed between the animals over the right thorax, an area containing relatively few blood cells. However, the signal over the liver was slightly enhanced in the mouse injected with the anti-CD4 antibody. A three- to fivefold higher signal was seen over the spleen and both cervical lymph nodes of the mouse injected with the anti-CD4 antibody in comparison to the control, highlighting the organs known to contain high densities of CD4 lymphocytes. Reproduced with permission from Nature Methods, vol. 3, no. 4, pp. 295–301 (2006).

the weakly complementing b-gal fragment were injected into nude mice(15). Upon agonist binding, b-arrestin2 bound to the activated GPCR, resulting in complementation of b-gal and an increase in enzyme activity (Fig. 20.3). Intraperitoneal injection of agonist and subsequent imaging using Lugal resulted in a robust luminescence induction, showing that b-gal complementation can be imaged and used to monitor GPCR activation in live animals. The experiments performed using Lugal have shown that b-gal can be imaged using bioluminescence. However, this is still a very recent technique, the optimization of numerous parameters is still ongoing and the ultimate value of the method as a research tool in

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Fig. 20.3. In vivo imaging of b2-adrenergic receptor activation using b-gal complementation in conjunction with sequential reporter-enzyme luminescence. Cells expressing the b2AR construct were transduced to express Fluc and K-Ras and injected in subcutaneous location into the back of BALB/c nude mice (4  106 cells/injection). (A) Seven to fourteen days later, when cells had grown into small tumors, baseline luminescence was imaged by injection of Lugal. Isoproterenol (6 mg/kg ip) or vehicle was injected and luminescence imaged again after 1, 8, 24, and 36 h. (B) Robust increase in luminescence was seen 1 h after isoproterenol injection, which subsequently returned to baseline within 24 h. (C) Quantification shows that signal increase was approximately fourfold over baseline (red line: mice treated with isoproterenol; blue line: vehicle-treated controls) (mean – SEM; n = 9/group). Reproduced with permission from FASEB J vol. 21, no. 14, pp. 3819–3826 (2007).

life sciences remains to be shown. The following technical description focuses on the general aspects of in vivo methods of b-gal imaging using Lugal. If other enzymes are to be imaged, a different set of tests might be required to establish the method.

2. Materials 2.1. Mice

1. Nude mice are, in general, best suited for optical imaging experiments because fur leads to partial extinction and scattering of the emitted light, leading to loss of signal and spatial resolution. BALB/c nude mice are available from several vendors, e.g., Taconic (Germantwon, NY) and Jackson (Bar Harbor, ME) and are especially well suited to this application because they also are T cell-deficient. Hence, immune response to bacterial b-gal, triggering a slight, localized accumulation of immune cells in immune-competent mice, can be avoided (unpublished observation).

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2. If nude mice cannot be used, mice with white or light fur are preferred. See Note 1. 3. Depending on the question addressed, the source of b-gal that is to be imaged is diverse, including engineered cells, gene transfer into adult animals, transgenic mice, and cells or antibodies labeled with recombinant b-gal protein. It would clearly go beyond the scope of this chapter to describe protocols for each experiment performed to date, which might be of interest to other researchers. 4. In the case of myoblasts retrovirally transduced with the LacZ and the Fluc genes, we refer the reader to the expert protocols published (16, 17). 2.2. Lugal

1. The substrate 1-O-galactopyranosyl-luciferin (Lugal) is commercially available from different sources. The authors have used the substrate obtained as a special order from Promega (Madison, WI), but it is also available from Marker Gene Technologies (Eugene, OR). 2. Lugal is the core ingredient of the BetaGlo1 Kit, an assay system for b-gal quantification. Because the kit is designed to be used as a terminal measurement in cell culture, it contains detergents and other unspecified agents that lead to cell lysis. Hence, the kit cannot be directly used as sold on living cells or for injection into living animals. An alternative approach is described in the Notes section (Note 2).

2.3. Imaging Device

Systems for bioluminescent in vivo imaging that include a cabinet, a CCD camera, as well as data processing and storage software are commercially available from at least three companies. The authors have used the IVIS1-100 and IVIS1-Spectrum systems (Xenogen-Caliper Life Sciences, Hopkinton, MA), but a similar system (NightOWL II LB 983) is available from Berthold Technologies (Bad Wildbad, Germany). As an example, the IVIS1-100 consists of the following components: l A light-tight imaging chamber. l

A heated stage (to avoid cooling off of the anaesthetized animals). The stage is sized to hold five mice or three rats, an important prerequisite for ‘‘relatively high throughput’’ imaging. Included are gas anesthesia connections and a full gas anesthesia system (e.g., isofluorane).

l

A charged-coupled-device (CCD) camera (2048  2048 pixels) cryogenically cooled to –90C by a closed-cycle refrigeration unit to minimize electronic background and maximize sensitivity.

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The system is operated via a computer using the Living Image1 3.0 software. The software also serves to store the data, for image display and analysis.

3. Methods 1. Lugal can be easily dissolved in PBS, aliquoted and frozen for later use (Lugal stock). 2. Before use, an aliquot of the Lugal stock is thawed and diluted in PBS to a final volume of 100 mL per mouse. 3. The Lugal dose used in most of our experiments was between 0.1 and 0.2 mmol/kg body weight. 4. Mice are anesthetized. Injectable compounds (e.g., Ketamine/ Xylacine or Avertin) can be used. The authors, however, prefer inhalation anesthesia (e.g., isofluorane), because it allows prolonged imaging sessions if necessary without the need for reinjection (which would interrupt the imaging procedure and result in altered body position). 5. Anesthesia is induced in an induction chamber with 3–4% isofluorane. 6. Mice are weighed and placed into the imaging chamber, where anesthesia is maintained through isofluorane administered through a nose cone (typically 1.75%). The position of the animal is chosen according to the localization of the organ of interest. See Note 3. 7. Based on body weight, the total dose of Lugal is determined and diluted in PBS to a final volume of 100 mL per mouse, and filled into a 1 mL insulin-type syringe fitted with a 27–29-gauge needle. 8. For intraperitoneal injection of Lugal, mice are briefly removed from the nose cone. It is important to inject head down into the lower left quadrant of the abdomen to minimize the risk of injury of internal organs. See Note 4. 9. Optimal settings of sensitivity (‘‘binning’’) and duration of exposure need to be determined for each experiment. We have used exposures of up to 180 s in experiments with low luminescence. In other instances, e.g., if b-gal was imaged in transgenic mice ubiquitously overexpressing Fluc, luminescence generally was high and exposure, hence had to be short (few seconds). Note: Binning causes reduction in spatial resolution, but this does not limit the quantitative readout of the signal.

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10. Collect serial images until the peak signal is achieved, typically after 20–30 min. See Note 5. 11. Images are stored by the software (e.g., Living Image1 3.0) for later analysis. Luminescence is represented by a color map over a gray-scale picture of the mouse and can be easily quantified by drawing regions of interest.

4. Notes 1. In some instances (e.g., specific transgenic or knockout mice), black mice have to be used instead of BALB/c nude or white mice. Luminescence and spatial resolution can be improved by shaving the mice over the region of interest if needed. 2. As an alternative to Lugal, the solid component (‘‘cake’’) of the BetaGlo1 Kit can be used, although its precise composition and, in particular, the concentration of Lugal contained are not disclosed by the manufacturer. In this case, the ‘‘cake’’ is not dissolved in the liquid buffer provided in the kit (which contains detergents), but rather in PBS. A variety of doses were used for in vivo experiments. In theory, the use of the solid component of the BetaGlo1 kit might provide an advantage because it also contains active firefly luciferase, ATP, and various inorganic salts, which may locally ‘‘burn off’’ any free Dluciferin contaminating the solution that would contribute to background luminescence. Whether this protocol reduces background, as expected, has not yet been established. 3. Anesthesia can be tricky in the setting of in vivo imaging, because the animals cannot be continuously surveyed while the images are acquired. It is recommended to regularly check on the animals every couple of minutes in between the acquisition of images. 4. Lugal, like the standard firefly luciferase substrate D-luciferin, is readily resorbed following intraperitoneal injection. This may sound like a detail but, in practice, dramatically reduces the requirements on time and technical skills. Indeed, it is not possible for a single operator to intravenously inject five mice simultaneously, because this technique typically takes up to several minutes per mouse. In contrast, five anesthetized mice can be easily injected intraperitoneally within a few seconds (‘‘simultaneously’’), placed in the imager and imaged. Thus, the option of intraperitoneal injection of the substrate is a prerequisite for ‘‘relatively high-throughput’’ imaging (if two imagers are available, up to 30 mice can be imaged per hour by a single operator). Similar to Lugal, the caspase-3-substrate

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(Caspase-Glo, Promega, Madison, WI) can be delivered intraperitoneally (12). If other conjugates are to be used, it will be necessary to determine first whether they are absorbed following intraperitoneal administration (as is the case for D-Luciferin and Lugal) or need to be injected intravenously. 5. Importantly, background luminescence was seen in all experiments, most likely due to ‘‘spillover’’ of free D-luciferin. Indeed, in the presence of active firefly luciferase (e.g., in the transplanted cells) that acts as the ‘‘helper enzyme,’’ any free luciferin will produce light. Lugal appears to have limited stability in mouse in plasma and, furthermore, to be partially degraded during freeze/thaw cycles, generating free luciferin. This problem can be circumvented in part by using exclusively images acquired within the first few minutes following Lugal injection, thus enabling reliable imaging of b-gal activity. In later images background luminescence was found to increase, apparently due to the generation of free luciferin, until, eventually, b-gal activity was no longer distinguishable at all. An additional reason for unspecific luminescent signal might be the presence of endogenous, mammalian b-gal, leading to cleavage of Lugal; such signal might be reduced or prevented if alternative models such as the b-gal knockout mice were used, or by using other reporter enzymes that are not unspecifically expressed in mammalians. Further studies are necessary to establish the stability of Lugal and to optimize the protocol. Chemical modification most likely could improve the stability in serum. If other substrates are to be used, this caveat needs to be cautiously monitored and characterized. References 1. Wu, J. C., Chen, I. Y., Wang, Y., Tseng, J R., Chhabra, A., Salek, M., Min, J. J., Fishbein, M. C., Crystal, R., and Gambhir, S. S. (2004) Molecular imaging of the kinetics of vascular endothelial growth factor gene expression in ischemic myocardium. Circulation 110, 685–691. 2. Contag, P. R., Olomu, I. N., Stevenson, D. K., and Contag, C. H. (1998) Bioluminescent indicators in living mammals. Nat Med 4, 245–247. 3. Contag, C. H., Contag, P. R., Mullins, J. I., Spilman, S. D., Stevenson, D. K., and Benaron, D. A. (1995) Photonic detection of bacterial pathogens in living hosts. Mol Microbiol 18, 593–603. 4. Edinger, M., Sweeney, T. J., Tucker, A. A., Olomu, A. B., Negrin, R. S., and Contag, C. H. (1999) Noninvasive assessment of

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tumor cell proliferation in animal models. Neoplasia 1, 303–310. Gross, S., and Piwnica-Worms, D. (2005) Real-time imaging of ligand-induced IKK activation in intact cells and in living mice. Nat Methods 2, 607–614. Zhao, H., Doyle, T. C., Coquoz, O., Kalish, F., Rice, B. W., and Contag, C. H. (2005) Emission spectra of bioluminescent reporters and interaction with mammalian tissue determine the sensitivity of detection in vivo. J Biomed Opt 10, 41210. Wehrman, T. S., von Degenfeld, G., Krutzik, P. O., Nolan, G. P., and Blau, H. M. (2006) Luminescent imaging of beta-galactosidase activity in living subjects using sequential reporter-enzyme luminescence. Nat Methods 3, 295–301. Geiger, R., Schneider, E., Wallenfels, K., and Miska, W. (1992) A new ultrasensitive

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bioluminogenic enzyme substrate for betagalactosidase. Biol Chem Hoppe Seyler 373, 1187–1191. Miska, W., and Geiger, R. (1987) Synthesis and characterization of luciferin derivatives for use in bioluminescence enhanced enzyme immunoassays. New ultrasensitive detection systems for enzyme immunoassays, I. J Clin Chem Clin Biochem 25, 23–30. Miska, W., and Geiger, R. (1988) A new type of ultrasensitive bioluminogenic enzyme substrates. I. Enzyme substrates with D-luciferin as leaving group. Biol Chem Hoppe Seyler 369, 407–11. Cali, J. J., Ma, D., Sobol, M., Simpson, D. J., Frackman, S., Good, T. D., Daily, W. J., and Liu, D. (2006) Luminogenic cytochrome P450 assays. Expert Opin Drug Metab Toxicol 2, 629–645. Shah, K., Tung, C. H., Breakefield, X. O., and Weissleder, R. (2005) In vivo imaging of S-TRAIL-mediated tumor regression and apoptosis. Mol Ther 11, 926–931.

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13. Monsees, T., Miska, W., and Geiger, R. (1994) Synthesis and characterization of a bioluminogenic substrate for alpha-chymotrypsin. Anal Biochem 221, 329–334. 14. Geiger, R., and Miska, W. (1989) A new type of ultrasensitive bioluminescence enzyme substrates for kininases. Adv Exp Med Biol 247B, 383–388. 15. von Degenfeld, G., Wehrman, T. S., Hammer, M. M., and Blau, H. M. (2007) A universal technology for monitoring G-protein-coupled receptor activation in vitro and noninvasively in live animals. Faseb J 21, 3819–3826. 16. Banfi, A., Springer, M. L., and Blau, H. M. (2002) Myoblast-mediated gene transfer for therapeutic angiogenesis. Methods Enzymol 346, 145–157. 17. Springer, M. L., Rando, T. A., Blau, H. M., Banfi, A., Springer, M. L., and Blau, H. M. (2002) Gene delivery to muscle. Myoblastmediated gene transfer for therapeutic angiogenesis. Curr Protoc Hum Genet Chapter 13, Unit 13 4.

INDEX The letters ‘f ’, ‘t’ and ‘n’ following locators refer to figures, tables and note numbers respectively.

A Accuracy................................................................ 19, 33–34 Acquisition..........4, 11 n3, 40, 41, 100, 102, 118, 119, 120, 121, 126, 128, 129, 131, 132f, 134 n6, 158, 163, 169, 200, 257 n3 Activity................................... 2f, 3, 4, 7, 11 n2, 13, 20, 27, 30f, 35 n12, 61 n4, 89, 90, 91, 102 n10, 105–106, 109, 111, 113f, 114f, 115–116, 118f, 121 n16, 122 n18, 123f, 128–129, 134 n2, 138, 141, 186f, 195, 196, 199 n1, 200 n9, 201 n15, 220, 249–258 Adenosine tri-phosphate .................................................. 25 Aequorea victoria.................................................................. 7 Aequorin/Aequorins ... 7–8, 203, 204–209, 210f, 211f, 212t Aequorea victoria............................................................ 7 Apoaequorin ..................................................... 7–8, 208 Analysis environmental ............................................................... 2 food ........................................................................... 247 multiplex ................................................................. 9–11 Anesthesia.............40, 41, 42, 43, 44 n5, 65, 66–67, 79, 81, 82, 109, 112, 117, 120 n14, 126, 134 n7, 197, 201 n12, 255, 256, 257 n3 Animal model ....................................15, 20, 116, 119, 125, 126 tranplantation.............................................................. 76 Anterior pituitary, see Pituitary Antibody ......... 31, 33, 35, 50, 102, 199, 200 n6, 239f, 240, 242, 243, 244, 246 n5, 247 n11, 253f Apoaequorin ........................................................... 7–8, 208 Apoprotein.....................................................................7, 8f Apoptosis ...................................... 1, 88, 105–114, 193–194 Area under the curve (AUC) ...............39–40, 178, 182 n15 Assay ..... 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 17–18, 20, 33–34, 35 n7, 36 n14, 88, 99, 108, 110, 111, 116, 120 n1, 122 n18, 126, 128–129, 132–133, 140–141, 144, 148 n5, 151 n19, 157, 160, 162, 163–164, 169, 174, 175f, 176, 178f, 179, 180 n7, 181 n12, 182 n16, 185–191, 194, 195, 196, 200 n1, 215–233, 239f, 242, 243, 244, 255 Assay system ....... 3, 4, 9, 11 n3, 13 n9, 108, 122, 140–141, 147 n5, 148 n6, 188, 195, 255

ATP, see Adenosine tri-phosphate Attenuation............16–17, 18–19, 20, 65, 116, 132, 134 n1 AUC, see Area under the curve (AUC) Autoinducer ........................................................................ 5

B Background ..... 9, 39, 42f, 43, 48, 57, 59–60, 65, 67, 70 n3, 76, 83, 88, 89, 115, 119, 121 n16, 128, 129, 130, 147 n5, 151 n19, 169, 177, 181 n14, 182 n15, 197, 209, 211, 222, 224f, 225f, 232 n21, 239f, 250, 255, 257 n2, 258 n5 Bacteria/Bacterial...2, 3, 4–5, 6, 7, 9, 10, 11, 12, 16, 27, 28, 109, 116, 137–151, 188, 207f, 250, 251, 254 colonization....................................................... 137–151 light emitting .................................................... 137–151 Bandwidth............................................................... 9, 10, 63 Bimolecular fluorescence complementation .......... 216, 217, 218, 220, 221, 222, 224, 225, 231 n15 Bioanalytical........................................................................ 1 Bioluminescence Imaging (BLI), see Imaging Bioluminescence Resonance Energy Transfer............... 157, 173–182, 215–233 Biosafety.................................................................. 133, 134 Biotin .......................................................................28, 253f Blocking .............................................................. 31, 33, 199 BRET, see Bioluminescence Resonance Energy Transfer Bright-GloTM Assay System ............3, 4, 11 n3, 13 n9, 148 Burden......................................... 15–16, 17–18, 37–45, 139

C Caged Luciferin .............................................................. 250 Calcium.....................7, 77, 78, 95, 203–213, 239, 240, 244 calcium-activated .......................................................... 7 intracellular or subcellular............................. 7, 203, 209 oscillation .......................................................... 203–213 Calibration .......................... 9, 30f, 32, 33–34, 40, 91f, 157, 158, 163–164, 166f, 180 n3, 204, 208f, 209, 212, 213 n7 Cancer ......... 15–16, 26, 37, 47, 48f, 56, 105, 156–157, 185 Caspase............105–106, 108, 109, 110, 111, 112, 113 n10, 114f, 250, 251, 257 n4 CCD, see Charge-coupled device (CCD) camera

P.B. Rich, C. Douillet (eds.), Bioluminescence, Methods in Molecular Biology 574, DOI 10.1007/978-1-60327-321-3, ª Humana Press, a part of Springer Science+Business Media, LLC 2009

261

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262 Index

Cell culture ....................3, 4, 5, 7, 10, 11, 27, 31, 33, 50–51, 95, 96, 97, 99, 108, 110, 128, 129, 131, 156, 174, 176, 188, 189, 195, 196, 207, 217, 220, 239, 250, 255 death...................................... 88, 105 See also Apoptosis differentiation ....................................................... 26, 88 engraftment........................................................... 15–16 line...................2, 3, 38, 56, 92, 128, 174, 204, 212, 214 lysis........................................................3, 4, 6, 204, 255 proliferation .......................................26, 90, 91f, 92, 93 stem cell, see Embryonic stem cell surface ..... 26, 28, 29f, 31, 33, 34, 35, 48, 49, 215, 237f, 239f, 244, 247 survival .......................................................... 15, 87–102 tracking ........................................................... 88–89, 90 transfection.............................................................. 3, 95 transplantation ............................................................ 18 Centrifugation ...............4, 12 n9, 56, 59, 96, 206, 243, 245 Charge-coupled device (CCD) camera ..........40, 41, 44 n4, 66, 67, 84 n5, 88, 89, 112, 114f, 115–116, 117, 120 n11, 126, 130, 131, 146 n1, 156, 162, 195, 197–198, 200 n9, 205, 209, 255 Circulation .............................. 41, 63, 66, 69f, 70 n3, 71 n6 Click Beetle........................................................................2f CMV, see Cytomegalovirus Coelenterazine ........5–6, 7, 12 n7, 110, 116, 126, 127, 128, 129, 130, 134 n5, 138, 141, 142, 143, 144, 148 n6, 151 n19, 173–174, 175, 176, 177, 180 n1, 181 n8, 190, 203–205, 208, 209, 212t, 218, 219, 220, 221, 222, 224, 226, 227f, 228f, 229, 230 n5 Collagenase ...................77, 78, 79–80, 94, 98, 99, 204, 206 Colonization ................................................... 119, 137–152 Confocal microscopy....................................................... 176 Conformational....................................................... 1, 7, 178 Conjugation/Conjugated........48, 50, 55, 56, 58, 61 n3, 64, 102 n11, 195, 239f, 240, 242, 243, 244, 246 n11, 250, 251, 253f, 258 n4 Culture .........2f, 3, 4, 5, 6, 7, 10, 11, 12 n9, 27, 28, 29, 30f, 31, 33, 34, 35 n8, 50, 56, 59, 81, 93, 94, 95, 96, 97, 98, 99, 101 n2, 108, 110–111, 112, 127, 128, 129, 131, 156, 174, 176, 177, 188, 189–190, 191f, 195, 196, 204, 205–206, 207, 212 n1, 217, 220, 239, 240, 241, 242, 243, 244–245, 247 n15, 250, 255 Cypridina noctiluca............................................................... 6 Cytomegalovirus .................. 92, 95, 109, 188, 190 n1, 236f

D Data acquisition ........................................................ 40, 126 Death .........................................88, 105, 139, 145, 201 n12 Deep Blue C ......................................................220, 230 n5 Degree of labeling............................................................. 55

Detection dual-color ...................................................................... 9 high sensitivity ..........................................2, 8, 129, 204 high throughput..................................2, 11, 12, 90, 179 in vivo ....................................................................... 251 limit of detection......................157, 158, 166, 167f, 169 non-invasive ........................... 1, 20, 90, 118f, 122f, 250 off-line .................................................................. 27, 31 See also Imaging Diabetes .......................................................... 75–76, 79, 88 Differentiation ..............1, 26, 87–88, 90–91, 193–194, 251 DOL, see Degree of labeling Dosing............134 n3, 147 n4, 149 n9, 150 n12, 156, 158f, 159, 160–161, 162, 163, 164, 165, 166, 167, 168 Dots............................................................................. 63–71 Dual color ........................................................................... 9 Dual-GloTM assay system................................................... 9 Dynamic range................... 2, 39f, 158f, 163, 169, 203–204

E Ecto-ATPase ......................................................30f, 35 n13 Efficiency ..........4, 92, 99, 112, 118, 120 n9, 156–157, 166, 180 n5, 189–190, 213 n7, 230 n10 Electroporation .............. 92, 158, 159, 161, 166f, 167f, 168 Elution ...............................................................9, 10, 12 n9 Embryonic Stem Cell ............................................... 87–102 Emission ............ 2f, 5, 6, 8, 9–10, 11 n3, 12 n8, 16, 18, 38, 39–40, 41, 43, 49f, 57, 59, 63, 65, 67, 68, 71 n5, 83 n3, 84 n5, 88, 89, 118, 119, 120 n7, 121 n16, 122 n18, 127, 128t, 129, 144, 145f, 146 n1, 147 n2, 151 n18, 156–157, 163, 169, 173–174, 175f, 177, 179, 181 n14, 182 n15, 198f, 203–204, 205, 208, 209, 210f, 211, 213 n7, 218, 219, 220, 221, 222, 225, 226, 227f, 228f, 229, 232 n20, 238f, 247 n11 Emitter...............................................................9, 11, 71 n5 Engraftment.............................................. 15–16, 75–76, 88 Epitope............................ 216, 236, 237f, 238, 239f, 246 n6 ESC, see Embryonic Stem Cell Exposure ........... 43, 44, 61 n1, 70 n1, 94, 97, 99, 118, 129, 138–139, 144f, 145f, 151 n21, 197, 198f, 200 n9, 247 n14, 256 Expression..... 1, 2, 3, 4, 5, 6, 10, 11 n3, 16, 20, 35 n11, 38, 90–91, 92, 101 n7, 108, 109, 110, 112, 115–116, 126, 127, 138–139, 142, 148 n5, 149 n11, 155–169, 174, 176, 179, 180 n5, 181 n14, 188, 194, 204, 205, 206, 207, 208, 213 n7, 221, 222, 223, 232, 236, 237f, 239, 241, 242, 243, 244, 246 n6, 247 n10, 250, 252 Extracellular .................25–36, 49, 105–106, 111, 119, 143, 144–146, 150 n15, 185, 187f, 215–216, 236, 237–238, 239f, 246 n3, 247 n10, 250, 251, 252, 253f Ex Vivo......................................16, 143, 145, 160, 163, 204

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Index 263

F

H

FACS ........88–89, 92, 99, 101 n7, 216, 240, 241, 243, 244, 245, 246 n4, 247 n11 Field of view........ 66, 82, 118, 129, 130, 131, 132f, 134 n6, 145, 197, 198f Firefly ...186–187, 188, 189–191, 191 n7, 249–250, 257 n2, 258 n5 Flow ...... 17, 43, 63–71, 82, 101 n7, 141, 163, 213 n5, 242, 244, 247 n12 Fluc ....... 91, 106, 107, 108, 109, 110, 112, 113f, 114f, 249, 250, 251, 252, 253f, 254f, 255, 256 Fluorescence.................................. 47–61, 227f, 228f, 237f, 238f, 239f BiFC, see Bimolecular fluorescence complementation GFP, see Green Fluorescent Protein protein................................................................... 55, 56 Fluorescence resonance energy transfer ........... 216, 217, 220, 226 Fluorophore ...................................................................... 49 FOV, see Field of view FRET, see Fluorescence Resonance Energy Transfer Fusion ..........90, 94, 95, 174, 176, 218, 219, 221, 222, 225, 226, 227f, 228f, 231 instrument..........................218, 219, 222, 225, 231 n17 protein....... 91, 174, 179 n6, 180 n5, 181 n12, 182 n14, 187f, 207, 212, 232 n22, 236

Herpes Simplex Virus .................... 90, 116, 122f, 123f, 186, 204, 206 hESC, see Embryonic Stem Cell High-sensitivity .............................................................. 129 High-throughput........................................................ 37–45 HSV, see Herpes Simplex Virus Hydrodynamic dosing............................................. 159, 165 Hypoxia............................................................................. 17

G b-Galactosidase............................................... 220, 249–258 Gaussia princeps ..........................................5, 126, 128t, 138 Gene...............2, 50, 66, 88, 92, 94, 95, 155–169, 174, 176, 185–191, 203, 217, 255 expression...........1, 2, 16, 138, 139, 155–169, 203, 244, 249, 252 knockdown........................................................ 155–169 non viral gene delivery ...................................... 155, 156 non viral gene expression .......................................... 155 regulation ...................................................................... 1 transgene ............................................... 4, 156, 161, 199 GFP, see Green Fluorescent Protein Gluc..................5, 6, 25, 29, 75, 76, 94, 137, 138, 140, 141, 142, 143, 147 n2, 148 n6, 160, 164, 165, 166, 181 n10, 205, 206, 209, 217, 240 GPCRs, see G protein-coupled receptors G protein-coupled receptors........................... 216, 252, 253 Green Fluorescent Protein .......... 88, 91f, 101 n7, 102 n11, 127, 176, 180 n1, 181 n11, 207f, 208, 209, 212 n3, 213 n3, 216, 217, 220, 221, 231 n16, 236, 237, 238, 241, 242, 245, 247 n16 Growth.......... 5, 7, 12 n5, 15, 38, 49, 61 n4, 65, 93, 94, 95, 99, 111, 127, 128, 138, 151 n19, 186, 188, 190, 191, 194, 236

I Imaging ..........1, 2, 8, 15–20, 37–44, 47–61, 63–71, 75–84, 87–102, 115–123, 125–134, 139, 140, 141, 142, 143, 145, 146 n1, 147 n7, 149 n11, 151 n21, 155–169, 185–191, 194, 195, 196, 197, 198f, 199 n10, 201 n10, 203–213, 216, 217, 235–247, 249–258 bioluminescence imaging (BLI) ........ 15, 37–44, 76, 79, 82, 91f, 92, 99, 100, 101 n8, 112f, 117, 118f, 123f, 125–134, 139, 141, 143, 156, 194, 195, 196, 197, 198f, 203–213, 249, 251 dual-color ...................................................................... 9 high sensitivity .......................................................... 129 in vivo ............................................... 169, 204, 249–258 molecular............................................................... 47, 88 multiplexed.................................................................... 8 non-invasive .................................................................. 1 optical........................................................................ 254 overlay ......................................................................... 41 plate........................................................................... 128 processing images........................................................ 42 real-time...................................................................... 76 single cell....................................................................... 2 spectral ........................................................................ 57 system........ 41, 44, 51, 57, 59, 70, 84, 90f, 101 n8, 117, 120 n12, 141, 158f, 160 well ..................................................2, 99, 243, 250, 255 See also Detection Immunofluorescence............................... 194, 195, 198, 240 Immunostaining.................................................200 n6, 241 Infection/Infectious ........................................ 115–123, 206 bacterial ................................................................... 5, 10 viral.................................................................... 125, 128 Inflammation .........................................1, 17, 26, 198f, 215 Infra-red...................................................................... 63, 66 Injection .........6, 7, 8, 19, 32, 38, 41, 43, 44, 51, 57, 59, 61, 65, 67, 68, 70, 78, 79, 81, 82, 83, 84, 91f, 93, 100, 101, 114f, 116, 117, 119, 121f, 127, 128, 130, 134, 140, 147, 148, 149, 150, 162, 168, 169, 177, 197, 198f, 200, 201, 251, 252, 253f, 254f, 255, 256, 257 n4, 258 n5 In situ ................................................................ 19, 147, 149

BIOLUMINESCENCE

264 Index

Instrument ........... 2, 3, 39, 42, 44, 77, 78, 81, 88, 102 n11, 129, 130, 131, 133, 134, 146 n2, 174, 177, 180, 190, 195, 205, 218, 219, 221, 222, 225, 231 n17 Integration .............. 5, 8, 10, 33, 38, 39, 41, 92, 118, 144f, 145f, 146f Intein............................................... 106, 107, 109, 185–191 Interference ............................................9, 35, 41, 66, 67 n4 Intracellular.... 2, 7, 12, 143, 203–213, 236, 237f, 239f, 252 Intramuscular dosing ...................................... 159, 161, 168 Intraperitoneal ......... 19, 57f, 79, 100, 114f, 115, 121f, 127, 128, 140, 141, 142, 143t, 149, 157, 251, 252, 253, 256, 257, 258 Intra-portal ........................................................... 78, 81–82 Intravenous .... 38, 116, 127, 130, 142f, 143f, 150 n12, 251, 257 n4, 258 In vivo ..................3, 7, 8, 16, 20, 37, 49, 63, 64, 70, 71, 76, 79, 84, 88, 89, 90, 91, 92, 93, 110, 111, 116, 122, 126, 127, 130, 133, 139, 140, 145, 148, 150, 151, 161, 165f, 169 n6, 204, 251, 252, 253f, 255, 257 n2 Islet..................................76, 77, 78, 79, 81, 83, 84, 87, 207 isolation....................................................................... 77 transgenic .................................................................... 76 transplantation ......... 16, 17, 18, 75, 76, 78, 81, 82f, 83, 84, 88, 90, 91f, 92, 99 Isolation .......................................................... 75, 77, 78, 79

K Knockdown ............................................................. 155–169

L Labeling .........10, 49, 55, 58, 61, 63, 64, 66, 68, 69, 70, 92, 200 n6, 219, 226, 227f, 228f, 232 n20, 239f, 247 LacZ................................................................ 251, 252, 255 Langerhans.............................................................. 204, 206 See also Islet Lectin .......................................................28, 35 n11, 49, 50 Lentivirus ...................................................... 92, 95, 96, 244 Light ..... 4, 5, 6, 7, 10, 11 n4, 12 n9, 16, 17, 18, 19, 30, 32, 33, 38, 39, 40, 41, 42f, 43, 44, 52, 56, 57, 58f, 60f, 66, 67, 83, 84, 88, 89 n6, 110, 116, 119, 120 n11, 121 n16, 122 n18, 126, 127, 130, 131, 132, 134 n3, 137–151, 157, 160, 162, 173, 174, 175, 177, 181, 190, 196, 199, 200 n4, 203, 205, 208, 209, 212 n3, 213 n6, 218, 219, 221, 222, 224, 225, 229, 230 n6, 232 n21, 238, 246, 250, 253f, 254, 255, 258 n5 box............................................................................... 41 detection...................................................................... 38 emission ....... 5, 18, 38, 39, 41, 43, 84 n5, 89, 119, 121, 122, 144f, 146 n1, 147 n2, 151 n18, 173, 175f, 177, 203, 208 integration..................................................................... 5 propagation ................................................................. 18

quantification ............................................................ 110 source .............................................................. 16, 18, 19 Limit of detection..................................157, 158, 167f, 169 Line (cell)..................................................2, 3, 56, 128, 244 Luciferase....... 3–13, 16, 17, 18, 20, 27, 28, 29f, 30, 31, 32, 33, 34, 35, 36, 38, 76, 82, 83, 84f, 89, 90, 91f, 93, 101 n9, 106, 108, 110, 111, 115, 116, 117, 118, 119, 120 n11, 121f, 122 n18, 126, 127, 128t, 129, 130, 132f, 133, 138, 139, 140, 141, 142, 147 n2, 148 n5, 149 n11, 156, 157, 158, 158f, 159, 160, 161, 162, 163, 164, 165f, 166f, 167f, 169, 176, 179 n1, 186, 187, 188, 189, 190, 191, 194, 195, 196, 199 n1, 200 n10, 201 n15, 212 n3, 217, 218, 220, 224, 227f, 228f, 229, 232 n20, 249, 250, 252, 257 n2, 258 n8 activity........... 4, 7, 20, 30f, 35 n12, 118, 119, 122, 129, 196, 199 n1, 201 n15 bacterial (Lux)....................................................... 5, 138 Bright-GloTM assay system .....3, 4, 9, 11, 13, 108, 122, 148 n6, 188, 195, 255 cell-surface ...........................................................27f, 28 click-beetle ...........................................................2f, 189 Cypridina noctiluca.....................................2f, 6, 7, 12 n7 Dual-GloTM assay sytem .............................................. 9 firefly (Fluc) .......... 2, 4, 6, 9, 18, 34 n1, 76, 88, 89, 91f, 101 n9, 106, 107, 108, 111, 115, 118f, 126, 127, 128, 130, 132f, 133, 138, 142, 147 n5, 160, 186, 187, 188, 189, 190, 191, 249, 250, 257 n2, 258 n5 Gaussia princeps (Gluc).............................................. 126 green............................................................................ 10 Photinus Pyralis ......................................... 106, 115, 159 Photorhabdus luminescens ........................................... 138 red ....................................................................... 8, 9, 10 Renilla reniformis (LR) (Rluc) ........................... 185, 187 reporter plasmid ............................................................ 3 soluble ............................................................28, 29f, 34 Staphylococcus protein A (SPA)-luciferase............. 31, 33 Steady-GloTM assay system...................................11 n3 Luciferin........ 4, 5, 6, 7, 9, 10, 11 n4, 12 n7, 17, 19, 27, 30, 31, 32, 33, 34, 35 n14, 38, 39, 40, 41, 43 n6, 79, 82, 83 n3, 84f, 88, 89, 99, 100, 102 n10, 109, 110, 111, 112, 115, 117, 118, 119, 120 n12, 121 n17, 126, 127, 128, 129, 130, 134, 137, 138, 140, 141, 142, 143, 144, 148 n5, 149 n11, 156, 157, 158, 159, 162, 163, 164, 166f, 169 n6, 189, 195, 196, 197, 198f, 200, 201, 250, 253f, 257 n4 caged luciferin ........................................................... 250 oxyluciferin.........................................17, 34 n1, 89, 138 Lugal ......................... 250, 251, 252f, 253f, 254f, 255, 256, 257 n2, 258 n4 Luminescence .................. 5, 6, 7, 10, 18, 30f, 32f, 84f, 122, 211, 249–258 sequential reporter enzyme luminescence (SRL) .................................................... 249–258

BIOLUMINESCENCE

Index 265

Luminometer ......................... 9, 27f, 140, 146 n2, 188, 195 Lux .............................................................. 5, 138, 140, 142 Lymph/Lymphatic...................................................... 63–71 Lysis .....3, 4, 6, 12, 108, 110, 111, 113, 122, 160, 164, 188, 190, 191, 196, 200, 204, 209, 212t, 255

M Magnetic Resonance Imaging (MRI) .............................. 92 MAPK, see Mitogen Activated Protein Kinase Miniaturization................................................................... 2 Mitogen Activated Protein Kinase......................... 185–191 Molecular ....... 5, 7, 8f, 16, 26, 56, 63, 65, 70 n2, 71 n4, 79, 88, 102 n11, 116, 175, 181 n8, 186, 195, 204, 216, 218, 219, 240, 245 biology............................................................... 7, 37, 88 imaging ....................................................................... 88 Monitoring........ 2, 6, 89, 92, 93, 106, 109, 118f, 119, 122f, 123f, 137–151, 156, 208 Mouse ......... 19, 31, 35 n11, 39f, 40, 41, 43, 50, 51, 56, 57, 58, 59, 60f, 61 n4, 64, 65, 68, 69, 76, 77, 79, 81, 82f, 84f, 90, 94, 96, 99, 100, 101 n2, 107, 112, 117, 118, 119, 120 n7, 125, 130, 132f, 157, 159, 167, 168 n2, 194, 195, 197, 204, 240, 244, 253f, 257 MRI, see Magnetic Resonance Imaging (MRI) Multiplex/Multiplexed ................................................. 8–11

N Non invasive................................. 1, 20, 90, 118f, 122f, 250

O Off line.................................................................27, 28f, 31 Optical ...............16–20, 66, 70, 89, 127, 140, 146 n1, 151, 194, 246 n9 Oscillation............................................................... 203–213 Overlay.......................................................41, 42f, 145, 163 Oxygen ........... 5, 7, 17, 20, 40, 41, 43, 65, 66, 84, 120 n12, 138, 141, 143, 149 Oxyluciferin .................................................. 17, 34, 89, 138

P Pancreas ........................................................56, 77, 79, 206 See also Islet Pathogen ....42, 116, 117, 119, 120, 128, 129, 144, 150 n15 PET, see Positron emission tomography pH ................. 4, 5, 7, 9, 10, 12, 29, 30, 35 n12, 40, 47–61, 63–71, 78, 93, 108, 138, 148, 160, 188, 195, 205, 217, 219, 229 n2 Pharmacokinetics ..............................17, 18, 19, 20, 38, 251 Phosphorylation .................................................... 1, 26, 193 Photinus Pyralis .............1, 3, 17, 30, 38, 89, 106, 115, 126, 127, 128t, 159, 189 Photoactivation ............................................... 235, 238, 245

Photobleaching ......................................................... 63, 238 Photon..........8, 16, 17, 19, 34, 43, 83, 84, 88, 89, 100, 111, 112, 116, 119, 120, 121f, 122f, 123f, 129, 132, 133, 139, 140, 144, 145f, 146, 147 n2, 150 n15, 151 n21, 156, 157, 158f, 163, 165, 166, 167f, 169, 174, 188, 197, 200, 204, 205, 209, 210, 211, 213, 252f, 253f Photoprotein ....................................................................... 7 calcium-activated .......................................................... 7 Photorhabdus luminescens ......................................... 138, 142 PIN-G reporter....................................................... 235–247 Pituitary .........................................................204, 205, 206f Plasmid..............38, 95, 112, 157, 159, 213, 222, 236, 240, 245, 247 Positron emission tomography ......................................... 88 Probe ................48f, 49f, 56, 57, 58f, 60f, 89, 99, 100, 157, 196, 218 Progression............................1, 61, 116, 119, 125, 129, 185 Proliferation ........................................ 26, 87–102, 185, 193 Promoter .......... 2f, 6, 76, 83 n1, 88, 92, 95, 106f, 109, 113, 126, 127, 132f, 157, 159, 160, 187f, 188, 189, 190, 194, 207f, 236f SBE promoter........................................................... 194 Propagation..................................................16, 18, 19, 207f Protein......1, 2, 4, 6, 7, 49, 55, 88, 147, 156, 173–182, 186, 188, 206, 212 n3, 217, 222, 231 n16, n18, 235, 236, 238, 252 A: Protein, see Staphylococcus protein A (SPA) apoprotein ................................................................7, 8f conformational changes ............................................ 1, 7 fluorescent..................... 88, 89, 91f, 92, 127, 176, 179, 207f, 236 fusion protein.......... 97, 174, 179, 180, 181, 182, 187f, 232, 236 intracellular ........................................................... 2, 236 membrane protein............................................. 236, 252 phosphorylation .................................................... 1, 193 photoprotein ................................................................. 7 post-translational modification................................. 186 protein-protein interactions..1, 157, 173–182, 220, 252 purification.......................................................... 12, 216 secreted...................................................................... 156 splicing .............................................................106f, 186 surface ......................................................................... 49 trafficking.................................................................. 235 Purification ...............................12, 13, 31, 34, 80, 216, 217 Purinoceptor ..............................................................26, 30f

Q Quantification............4, 15, 16, 17, 18, 110, 116, 118, 119, 123f, 129, 131, 132, 139, 146f, 189, 211, 222, 231, 253f, 254f, 255 Quantum dots ............................................................. 63–71

BIOLUMINESCENCE

266 Index R

Raf-1 ....................................................................... 185–191 Ras..................................................................185–191, 254f Real-time .............. 6, 27f, 28, 29f, 31, 33, 60, 76, 111, 119, 125–134, 178f Region of interest ........ 41, 43, 129, 131, 132f, 197, 209 n1 Regression......................................................... 15, 164, 224 Regulatory DNA sequence.............................................................. 2 element.......................................................................... 1 Relative light unit ................................................... 110, 190 Renilla reniformis..................................... 108, 126, 187, 189 Reporter .........2, 3, 6, 7, 8, 10, 87–102, 108, 110, 116, 119, 126, 127, 128, 129, 130, 132, 133, 138, 156, 186, 188, 189, 194, 199 n1, 235–247 construct...................................................................... 92 gene ............................................................................... 2 intein-mediated................................................. 185–191 PIN-G reporter................................................. 235–247 plasmid.....................................................................2f, 3 single step reporter activity assay .................................. 3 system........................................................................ 251 Resolution ......10, 47, 82, 89, 118, 129, 131, 134, 151, 197, 254, 256, 257 Resonance ................................. 92, 157, 173–182, 215–232 See also BRET; FRET; MRI Reticuloendothelial system (RES)........................ 64, 66, 70 Rluc ..................89, 108, 110, 176, 178, 181, 182, 218, 219, 221, 222, 223f, 224, 225f, 226, 227f, 228f, 231, 232 n19 RLU, see Relative Light Unit RNA................................................................ 127, 157, 160 RNA interference (RNAi)................................ 155–169 shRNA .....................................157, 160, 164, 165f, 166 siRNA ............................................... 157, 160, 164, 165 ROI, see Region of interest

S Saccharomyces cerevisia ......................................................... 4 SBE promoter................................................................. 194 Second messenger ............................................................... 1 Sensitivity.........2, 4, 8, 12, 35, 36, 47, 55, 66, 83, 127, 129, 130, 131, 134 n2, 151 n21, 169, 197, 204, 209, 250, 255, 256 Sequential reporter enzyme luminescence (SRL) .. 249–258 shRNA ...........................................157, 160, 164, 165f, 166 Signal/Signaling (pathway)............................. 193, 194, 215 Single step................................................................... 3, 128 siRNA ............................................. 157, 160, 164–165, 166 Smad ....................................................................... 193–201 Small-volume sample analysis ............................................ 2 SNAP...... 217, 219, 221, 226, 227, 228, 232 n20, 232 n22, 232 n25

SPA, see Staphylococcus protein A Spectral/Spectrum...............................12, 39, 127, 161, 255 imaging ................................................................. 57, 59 resolution .............................................................. 10–11 spectral emission ......................................................... 18 Splicing ........................................................... 106, 109, 186 Staphylococcus protein A.........28, 31, 33, 34, 35 n10, 35 n11 Steady-GloTM assay system...................................... 11, 148 Stem cell, see Embryonic Stem Cell Substrate administration ................................................. 38, 79, 82 injection .................................................................... 255 preparation ................................................................ 219 System assay.........3, 4, 9, 11, 108, 122, 140, 148, 188, 195, 255 detection.................................................................... 250 reporter................................................................ 10, 251 reticuloendothelial ....................................64, 66, 68, 70

T Target.......2, 4, 8, 47–61, 63, 64, 65, 67, 88, 120, 161, 165, 167, 168, 187f, 204, 207f, 208, 212, 215, 216, 236f, 237f, 238, 250 Temperature....... 4, 8, 11 n2, 12 n6, 32, 35 n12, 52, 53, 54, 55, 58, 76, 78, 79, 80, 93, 95, 96, 98, 111, 113, 130, 139, 141, 168, 188, 198, 199, 201 n13, 208, 217, 219, 220, 221, 224, 229 n2, 242 TGFb, see Transforming Growth Factor b Tomography.................................................1, 18, 70 n1, 88 See also Positron emission tomography Tracking............................. 76, 88, 90, 91f, 92, 120 n8, 238 Trafficking .............................................. 131, 216, 235–248 Transcription ........................6, 88, 119, 186, 189, 194, 207 Transfection efficiency .................................................4, 92, 112, 189 transgene ............................................... 4, 156, 161, 199 Transforming Growth Factor b, 193–201 Transgenic/Transgene ....76, 83, 84, 194, 197, 199 n1, 251, 252, 253f, 255, 256, 257 n1 Transplantation.... 16, 17, 18, 75, 76, 78, 81, 82, 83 n1, 84, 88, 90, 91f, 92, 99 Tumor burden ....................................................... 15, 17, 37–45 growth ......................................................................... 15 progression .................................................................... 1 regression .................................................................... 15

U Ultrasensitive detection....................................................... 2

V Vaccinia........................................................... 131, 132, 133 Validation............................15–20, 127, 129, 133, 174, 176

BIOLUMINESCENCE

Index 267

Vasculature.................................................................. 63–71 Vector.........3, 6, 7, 88, 91, 92, 93, 105, 109, 110, 159, 160, 174, 176, 187, 189, 191, 195, 204, 206, 207, 225, 229, 236, 237, 240, 245 Venus ..............................................174, 176, 177, 180, 182 Virus Cytomegalovirus (CMV)...................... 92, 95, 109, 188 Herpes simplex virus (HSV)........90, 116, 118, 122, 123, 186, 204, 206 Lentivirus ...................................................... 92, 95, 244

W Wavelength....6, 9, 10, 12, 19, 65, 66, 68, 71, 89, 127, 173, 177, 178, 179, 182, 220

Y Yeast................................................................2, 3, 4, 5, 6, 7 See also Saccharomyces cerevisia YFP ...........................88, 180, 181, 216, 218, 222, 224, 225