Single-Cell Protein Analysis: Methods and Protocols (Methods in Molecular Biology, 2386) 1071617702, 9781071617700

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Methods in Molecular Biology 2386

Aik T. Ooi Editor

Single-Cell Protein Analysis Methods and Protocols

METHODS

IN

MOLECULAR BIOLOGY

Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, UK

For further volumes: http://www.springer.com/series/7651

For over 35 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-bystep fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitute the key ingredient in each and every volume of the Methods in Molecular Biology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexed in PubMed.

Single-Cell Protein Analysis Methods and Protocols

Edited by

Aik T. Ooi Mission Bio, Inc., South San Francisco, CA, USA

Editor Aik T. Ooi Mission Bio, Inc. South San Francisco, CA, USA

ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-0716-1770-0 ISBN 978-1-0716-1771-7 (eBook) https://doi.org/10.1007/978-1-0716-1771-7 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022, Corrected Publication 2022 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Cover Illustration Caption: Image provided by Valentina Proserpio. This Humana imprint is published by the registered company Springer Science+Business Media, LLC part of Springer Nature. The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A.

Preface Proteins are the most abundant type of macromolecules in a cell and are hugely diverse in their roles and functions. Important cellular processes, for example, the synthesis of DNA, RNA, and proteins, are catalyzed by enzymes, a type of catalytic proteins. The structural components of a cell are provided by cytoskeletal proteins. Some proteins are transport proteins located on the cell membrane that control the movement of a variety of ions, small molecules, and macromolecules in and out of the cell. Proteins are also involved in signal transduction pathways that regulate gene expression in response to certain stimuli. Since proteins are involved in a wide variety of important cellular processes, the detection and analysis of specific proteins in a cell could provide a deeper understanding of cell type and cell state. A large portion of molecular and cellular biology research efforts are devoted to exploring the presence, activity, localization, and function of specific proteins in a cell. Therefore, methods for protein analysis are required in various fields of research within molecular and cellular biology. In studies focusing on disease initiation and progression, identifying abnormal protein level and activity is crucial in the development of better diagnostics and therapies. In these biological samples, the cells within the samples could be extremely heterogeneous. Conventional protein analysis methods like enzyme-linked immunosorbent assay (ELISA) and Western blot require protein input amount from many cells and provide only an averaged result across all cells, hence not an accurate representation of the actual cells of interest. For these heterogeneous samples, a single-cell analysis method is required for a more truthful characterization of the cells of interest. While single-cell technologies have been rapidly evolving and improving in recent years, the majority of the single-cell methods available are for RNA sequencing. This is partly due to the easily available technology for amplifying nucleic acid by polymerase chain reaction (PCR), an important step to enhance the signals from the low amount of single-cell input material. For proteins, amplifying signals from single cells is not straightforward. Researchers are able to find ways, however, by combining different protocols, taking advantage of new emerging technologies, and improving upon conventional methods to perform protein analysis in single cells. This volume is a collection of methods for single-cell protein analysis. Each chapter provides detailed stepwise description of one protocol, with a “Notes” section devoted to authors’ insights and suggestions, additional recommendations, troubleshooting guides, and extra discussions. Readers will obtain the full protocols along with the invaluable experience shared by the authors. Several chapters in the volume are methods performed on tissue sections. Such methods might not be apparent as single-cell methods, but they do provide single-cell read out of the protein expression data; the ability to use archival tissues is also extremely beneficial in translational research. The methods in this volume range from simple to complex, conventional to the most current technologies. With the advancements in detection sensitivity, specificity, and throughput of the various tools employed, researchers can now analyze more than 40 proteins from a single cell in certain methods. More recently, researchers are finally able to perform multi-omic studies by obtaining protein expression data along with the analysis of RNA, DNA, or both in single cells. Readers can

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choose the method best suited for them based on their sample type and the technologies or equipment accessible to them. These protocols and the authors’ notes will be helpful for researchers hoping to utilize single-cell protein analysis in their studies. Since most of the new technologies are built upon improving existing approaches, the protocols collected here could also be the basis for the next generation of improved protein analysis methods in single cells. South San Francisco, CA, USA

Aik T. Ooi

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

1 Immunochemistry Analysis Using Chromogenic Substrates on Tissue Sections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Preethi Vijayaraj 2 Indirect Immunofluorescence of Tissue Sections . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cody J. Aros 3 Flow Cytometry for Beginners: Hints and Tips for Approaching the Very First Single-Cell Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Valentina Proserpio, Laura Conti, and Salvatore Oliviero 4 High-Dimensional Immunophenotyping with 37-Color Panel Using Full-Spectrum Cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Marco A. Fernandez, Hammad Alzayat, Maria C. Jaimes, Yacine Kharraz, Gerard Requena, and Pedro Mendez 5 Detection of Cytokine-Secreting Cells by Enzyme-Linked Immunospot (ELISpot). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bernt Axelsson 6 Detection and Enumeration of Cytokine-Secreting Cells by FluoroSpot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bernt Axelsson 7 Single-Cell Protein Profiling by Microdroplet Barcoding and Next-Generation Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Samuel C. Kim, John R. Haliburton, Zev J. Gartner, and Adam R. Abate 8 Single Cell Proteomics Using Multiplexed Isobaric Labeling for Mass Spectrometric Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ´ kos Ve´gva´ri, Jimmy E. Rodriguez, and Roman A. Zubarev A 9 Customizing Maxpar Direct Immune Profiling Assay with Additional Surface Marker and Intracellular Cytokine Staining Workflows for Expanded Mass Cytometry Panels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Carlene Petes, Stephen K. H. Li, Shariq Mujib, Michelle M. Poulin, Noah Saederup, Andrew A. Quong, and Christina Loh 10 High-Dimensional Tissue Profiling by Multiplexed Ion Beam Imaging. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ofer Elhanani, Leeat Keren, and Michael Angelo 11 Ultra-Sensitive Quantification of Protein and mRNA in Single Mammalian Cells with Digital PLA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jing Lin and Savas¸ Tay

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High-Throughput Multimodal Single-Cell Targeted DNA and Surface Protein Analysis Using the Mission Bio Tapestri Platform. . . . . . . . . . . . . . . . . . . . David W. Ruff, Dalia M. Dhingra, Kathryn Thompson, Jacqueline A. Marin, and Aik T. Ooi 13 Simultaneous Analysis of Single-Cell Transcriptomes and Cell Surface Protein Expression of Human Hematopoietic Stem Cells and Progenitors Using the 10x Genomics Platform . . . . . . . . . . . . . . . . . . . . Nicole Mende, Elisa Laurenti, Berthold Go¨ttgens, and Nicola K. Wilson 14 Generation of Centered Log-Ratio Normalized Antibody-Derived Tag Counts from Large Single-Cell Sequencing Datasets . . . . . . . . . . . . . . . . . . . . Benjamin Lacar 15 Simultaneous Quantification of Single-Cell Proteomes and Transcriptomes in Integrated Fluidic Circuits. . . . . . . . . . . . . . . . . . . . . . . . . . . Mandi Wong, Carol Kosman, Liane Takahashi, and Naveen Ramalingam 16 Combined Measurement of RNA and Protein Expression on a Single-Cell Level. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Valentina Russo, Nadia Brasu, and Luigia Pace 17 Simultaneous DNA, RNA, and Protein Analysis from Single Cells Using a High-Throughput Microfluidic Workflow for Resolution of Genotype-to-Phenotype Modalities. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dalia M. Dhingra, Aik T. Ooi, and David W. Ruff Correction to: High-Dimensional Immunophenotyping with 37-Color Panel Using Full-Spectrum Cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Contributors ADAM R. ABATE • Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA, USA HAMMAD ALZAYAT • Flow Cytometry Facility, Germans Trias i Pujol Research Institute, Badalona, Spain MICHAEL ANGELO • Department of Pathology, Stanford University, Stanford, CA, USA CODY J. AROS • UCLA Department of Molecular Biology Interdepartmental Program, UCLA, Los Angeles, CA, USA; UCLA Medical Scientist Training Program, David Geffen School of Medicine, UCLA, Los Angeles, CA, USA; UCLA Children’s Discovery and Innovation Institute, Mattel Children’s Hospital UCLA, Department of Pediatrics, David Geffen School of Medicine, UCLA, Los Angeles, CA, USA BERNT AXELSSON • Mabtech AB, Nacka Strand, Stockholm, Sweden NADIA BRASU • Armenise-Harvard Immune Regulation Unit, Italian Institute for Genomic Medicine (IIGM), Turin, Italy; Candiolo Cancer Institute, FPO-IRCCS, Turin, Italy LAURA CONTI • Molecular Biotechnology Center, University of Turin, Torino, Italy DALIA M. DHINGRA • Mission Bio, Inc., South San Francisco, CA, USA OFER ELHANANI • Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel MARCO A. FERNANDEZ • Flow Cytometry Facility, Germans Trias i Pujol Research Institute, Badalona, Spain ZEV J. GARTNER • Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA, USA BERTHOLD GO¨TTGENS • Department of Haematology, Wellcome and MRC Cambridge Stem Cell Institute, Jeffrey Cheah Biomedical Centre, University of Cambridge, Cambridge, UK JOHN R. HALIBURTON • Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA, USA MARIA C. JAIMES • Cytek Biosciences, Fremont, CA, USA LEEAT KEREN • Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel YACINE KHARRAZ • Cytek Biosciences, Fremont, CA, USA SAMUEL C. KIM • Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA, USA; Gilead Sciences, Foster City, CA, USA CAROL KOSMAN • Fluidigm Corporation, South San Francisco, CA, USA BENJAMIN LACAR • University of California, San Francisco, San Francisco, CA, USA; University of California, Berkeley, Berkeley, CA, USA ELISA LAURENTI • Department of Haematology, Wellcome and MRC Cambridge Stem Cell Institute, Jeffrey Cheah Biomedical Centre, University of Cambridge, Cambridge, UK STEPHEN K. H. LI • Fluidigm Canada Inc., Markham, ON, Canada JING LIN • Pritzker School of Molecular Engineering, University of Chicago, Chicago, IL, USA; Institute for Genomics and Systems Biology, University of Chicago, Chicago, IL, USA CHRISTINA LOH • Fluidigm Canada Inc., Markham, ON, Canada JACQUELINE A. MARIN • Mission Bio, Inc., South San Francisco, CA, USA

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NICOLE MENDE • Department of Haematology, Wellcome and MRC Cambridge Stem Cell Institute, Jeffrey Cheah Biomedical Centre, University of Cambridge, Cambridge, UK PEDRO MENDEZ • Synthego Corporation, Menlo Park, CA, USA SHARIQ MUJIB • Fluidigm Canada Inc., Markham, ON, Canada SALVATORE OLIVIERO • IIGM Foundation—Italian Institute for Genomic Medicine, Torino, Italy; Molecular Biotechnology Center, University of Turin, Torino, Italy; Dipartimento di ` degli studi di Torino, Torino, Italy Scienze della Vita e Biologia dei Sistemi, Universita AIK T. OOI • Mission Bio, Inc., South San Francisco, CA, USA LUIGIA PACE • Armenise-Harvard Immune Regulation Unit, Italian Institute for Genomic Medicine (IIGM), Turin, Italy; Candiolo Cancer Institute, FPO-IRCCS, Turin, Italy CARLENE PETES • Fluidigm Canada Inc., Markham, ON, Canada MICHELLE M. POULIN • Fluidigm Corporation, South San Francisco, CA, USA VALENTINA PROSERPIO • IIGM Foundation—Italian Institute for Genomic Medicine, Torino, Italy; Candiolo Cancer Institute, FPO-IRCCS, Torino, Italy ANDREW A. QUONG • Fluidigm Corporation, South San Francisco, CA, USA NAVEEN RAMALINGAM • Fluidigm Corporation, South San Francisco, CA, USA GERARD REQUENA • Flow Cytometry Facility, Germans Trias i Pujol Research Institute, Badalona, Spain JIMMY E. RODRIGUES • Division of Chemistry I, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden DAVID W. RUFF • Mission Bio, Inc., South San Francisco, CA, USA VALENTINA RUSSO • Armenise-Harvard Immune Regulation Unit, Italian Institute for Genomic Medicine (IIGM), Turin, Italy; Candiolo Cancer Institute, FPO-IRCCS, Turin, Italy NOAH SAEDERUP • Fluidigm Corporation, South San Francisco, CA, USA LIANE TAKAHASHI • Fluidigm Corporation, South San Francisco, CA, USA SAVAS¸ TAY • Pritzker School of Molecular Engineering, University of Chicago, Chicago, IL, USA; Institute for Genomics and Systems Biology, University of Chicago, Chicago, IL, USA KATHRYN THOMPSON • Mission Bio, Inc., South San Francisco, CA, USA ´ KOS VE´GVA´RI • Division of Chemistry I, Department of Medical Biochemistry and Biophysics, A Karolinska Institutet, Stockholm, Sweden PREETHI VIJAYARAJ • Immunology and Respiratory Research, Boehringer Ingelheim Pharmaceuticals Inc., Ridgefield, CT, USA NICOLA K. WILSON • Department of Haematology, Wellcome and MRC Cambridge Stem Cell Institute, Jeffrey Cheah Biomedical Centre, University of Cambridge, Cambridge, UK MANDI WONG • Fluidigm Corporation, South San Francisco, CA, USA ROMAN A. ZUBAREV • Division of Chemistry I, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden

Chapter 1 Immunochemistry Analysis Using Chromogenic Substrates on Tissue Sections Preethi Vijayaraj Abstract Immunohistochemistry (IHC) is a highly sensitive protein detection technique developed using the principle of antigen-antibody binding reaction. With immunohistochemistry, it is possible to visualize the abundance, distribution, and localization of proteins in situ. This chapter discusses the standard protocols involved in IHC detection using chromogenic substrates, including pre-treatment of tissues, types of chromogenic substrates, and troubleshooting at various stages of the protocol. Key words Immunohistochemistry, Chromogenic substrate, Fixation, Antigen retrieval, Permeabilization, Blocking, Detection, Troubleshooting

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Introduction Immunohistochemistry like immunofluorescence and immunocytochemistry makes use of antibodies to detect protein abundance, distribution, and localization within a cell or tissue. With specific protein levels being differentially regulated in health and disease, IHC is very commonly used in diagnostic and research laboratories. Detection of the protein is then facilitated by the use of enzyme labels, such as peroxidase [1] and alkaline phosphatase [2], colloidal gold [3], or radioactive elements [4] where the immunoreaction can be visualized by light microscopy, electron microscopy, or autoradiography, respectively. Estimation of the expression level of the protein is subjective and scored by trained personnel. Thus, while IHC provides superior spatial resolution, the outcome and success of the technique is experimenter dependent. This chapter focuses on principles, materials, and methods used in IHC using chromogenic detection methods. Additionally, strategies for standardization of staining and troubleshooting are discussed.

Aik T. Ooi (ed.), Single-Cell Protein Analysis: Methods and Protocols, Methods in Molecular Biology, vol. 2386, https://doi.org/10.1007/978-1-0716-1771-7_1, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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Materials

Tissue Fixation

Use of qualified reagents is of utmost importance to reproducibly perform IHC. Therefore, it is important to choose the best available grade reagents for this technique. Prepare reagents using deionized water and store at appropriate temperature as required. Use appropriate personal protective equipment and hazardous and toxic reagents should be disposed as labeled. 1. Dilution buffer: 1
Tris Buffered Saline (TBS). Dissolve 6.05 g of Tris, 9.76 g NaCl in 800 mL of H2O. Adjust pH to 7.4 with 1 M HCl and make up the volume to 1 L with H2O (see Note 1). 2. Wash Buffer: 1
Tris Buffered Saline—0.1% Tween (TBST). To 1 L of TBS, add 1 mL of Tween® 20. Mix well and store at room temperature for a maximum of 3 months. 3. 10% formalin (3.7% formaldehyde): use a chemical fume hood to dilute 37% formaldehyde 1:10 in dilution buffer. Prepare fresh each time (see Note 2).

2.2 Tissue Embedding, Sectioning, Deparaffinization, Rehydration, and/or Dehydration

1. Automated tissue embedder 2. Microtome 3. Positively charged slides with tissue sections 4. Heating block 5. Slide containers 6. Xylene (see Note 3) 7. 100% Ethanol 8. 90% Ethanol 9. 70% Ethanol 10. Deionized water 11. Toluene-based mounting medium (see Note 4) 12. Coverslips

2.3 Antigen Retrieval Solutions (see Note 5) 2.3.1 Heat-Induced Antigen Retrieval Solutions (HIER)

1. Sodium Citrate Buffer: 10 mM sodium citrate, 0.05% Tween 20, pH 6.0. Dissolve 2.94 g tri-sodium citrate (dihydrate) in 800 mL of water. Adjust pH to 6.0 with 1 N HCl and make volume up to 1 L with water. Add 0.5 mL of Tween 20 and mix well. Store at room temperature for 3 months or at 4  C for longer storage. 2. EDTA Buffer: 1 mM EDTA, 0.05% Tween 20, pH 8.0. Dissolve 0.37 g EDTA in 800 mL of water. Adjust pH to 8.0 using 1 N NaOH and make volume up to 1 L with water. Add 0.5 mL of Tween 20 and mix well. Store at room temperature for 3 months or at 4  C for longer storage.

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3. Tris-EDTA Buffer: 10 mM Tris Base, 1 mM EDTA Solution, 0.05% Tween 20, pH 9.0. Dissolve 1.21 g Tris Base and 0.37 g EDTA in 800 mL of water. Adjust pH to 9.0 using 1 N NaOH and make volume up to 1 L with water. Add 0.5 mL of Tween 20 and mix well. Store at room temperature for maximum of 3 months or at 4  C for longer storage. 2.3.2 Protease-Induced Antigen Retrieval (PIER)

1. Pepsin: 0.5% in 5 mM HCl pH 2.0. Dissolve 100 mg pepsin in 10 mL of 10 mM HCl (pH 2.0) to make 1% pepsin stock solution. Aliquot and store at 20  C. Dilute 1 mL of the pepsin stock solution in 1 mL of deionized water. Mix well and use immediately. 2. Trypsin (0.05%): Dissolve 50 mg trypsin in 10 mL water to make 0.5% trypsin stock solution. Aliquot and store at 20  C. Prepare 1% calcium chloride stock solution by dissolving 0.1 g calcium chloride in 10 mL water. Store at 4  C. Mix 1 mL of 0.5% trypsin stock solution and 1 mL of 1% calcium chloride stock solution in 8 mL of distilled water. Adjust pH to 7.8 with 1 N NaOH and use immediately.

2.4 Antigen Retrieval Equipment

1. Microwavable pressure cooker (e.g., Nordic Ware Microwave Tender Cooker 2.5 Quart or equivalent). 2. Heat-proof slide containers.

2.5 Tissue permeabilization and Blocking 2.5.1 Hydrophobic Pen: (e.g., Dako Pen)

2.5.2 Permeabilization Buffer (for Detection of Intracellular Antigens)

1. Harsh detergent: 0.1–0.2% Triton-X in dilution buffer

2.5.3 Endogenous Activity blocking (see Note 6)

1. Aqueous peroxidase blocking solution: 3% H2O2 in dilution buffer. Add 10 mL 30% H2O2 to 90 mL of dilution buffer, mix well, store at 4  C for up to 3 months (see Note 7).

2. Mild detergent: 0.2–0.5% of Tween 20, saponin, digitonin, or leucoperm in dilution buffer.

2. Alcoholic peroxidase blocking solution: 0.3% H2O2 in methanol. Add 1 mL of 30% H2O2 to 99 mL of methanol. Mix well and use immediately (see Note 7). 3. Universal blocking buffer: to 10 mL of dilution buffer, add 100 mg of bovine serum albumin, 10 mg cold fish skin gelatin, 50 μL Triton x—100, 5 μL sodium azide solution. Mix well and store at 4  C (see Note 8).

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4. Avidin blocking solution: 0.001% of avidin (10 U/mg) in dilution buffer. Store at 4  C (see Note 9). 5. Biotin blocking solution: 0.001% biotin in dilution buffer. Store at 4  C (see Note 9). 2.6 Chromogens and Substrates

1. 3,30 -Diaminobenzidine (DAB) peroxidase substrate solution: 0.05% DAB, 0.015% H2O2 in 0.01 M PBS, pH 7.2) (see Note 10). Add 250 μL 20X stock DAB (dissolve 0.1 g of 3,30 -diaminobenzidine, or DAB-tetrahydrocholoride in 10 mL deionized distilled water. Add 3–5 drops of 10 N HCl and mix well to dissolve. Aliquot the 20X stock DAB and store at 20  C) to 5 mL of dilution buffer and mix well. Add 250 μL of 20X stock H2O2 (dilute 100 μL of 30% H2O2 in 10 mL of deionized water and mix well. Use immediately. 2. 3-amino-9-ethylcarbazole (AEC) peroxidase substrate solution: 0.05% AEC and 0.015% H2O2 in 0.05 M acetate buffer, pH 5.5. Add 250 μL of 1 M acetate buffer (dissolve 13.6 g sodium acetate trihydrate in 100 mL of distilled water and adjust pH to 5.5 using concentrated HCl) to 5 mL of distilled water and mix well. Add 250 μL of 20X AEC (dissolve 0.1 g of AEC in 10 mL of 100% dimethyl formamide, store at 4  C) and mix well. Add 250 μL of 20X H2O2 and mix well. Use immediately (see Note 11). 3. New Fuchsin alkaline phosphatase substrate solution: 0.01% New Fuchsin, 0.02% sodium nitrite, 0.05% Naphthol AS-BI phosphate, 1 mM Levamisole, in 0.05 M Tris-HCl, pH 8.7. Add 250 μL of 20X New Fuchsin (0.2% New Fuchsin in 2 N HCl) to 5 mL of 0.05 M Tris-HCl, pH 8.7 and mix well. Add 250 μL of 0.4% sodium nitrite, 20 mM Levamisole and mix well. Add 250 μL 1% Naphthol AS-BI phosphate and mix well (see Notes 6 and 11).

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Methods It is crucial that the tissue sections do not dry out at any time during the IHC process. Use of a humidified chamber to perform all the incubations will prevent the tissue sections from drying out (see Note 12). Drying at any stage will cause nonspecific binding and high background staining. Optimum dilution of the primary and secondary antibody may be listed on the datasheets or must be empirically determined by the user (see Note 24). Adhere strictly to all incubation times in the protocol (see Note 24). For enzymatic methods of IHC, horseradish peroxidase (HRP) or alkaline phosphatase (AP) are the most commonly used enzymes. This section will discuss the steps involved in IHC analysis (Fig. 1) with the use of DAB, AEC, and New Fuchsin as chromogens, which are commonly used substrates with these enzymes.

Immunochemistry on Tissue

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Tissue Fixation (12-24 hrs)

Tissue Embedding/sectioning (1-2 days)

Deparaffinization (30 minutes)

Antigen retrieval (5-30 minutes)

Permeabilization (5-30 minutes)

Blocking (30 minutes – 1 hour)

1o Antibody Incubation (over night)

2o Antibody Incubation (30 minutes – 1hour)

Signal Amplifcation (1-2 hours)

Staining (3-10 minutes)

Fig. 1 Flow chart illustrating the immunohistochemistry protocol. Suggested timing depicts only the incubation times and not the preparation time 3.1 Tissue Fixation and Processing

Tissue preparation is a crucial step for IHC analysis. To preserve tissue architecture, prompt and adequate fixation is required. Inappropriate, inadequate or prolonged fixation could affect the antibody binding capability. The fixative recommended depends on the antigen, antibody, and tissue type. The most commonly used fixative routinely used in IHC procedures is 10% formalin (see Notes 2, 13, and 24) [5].

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Perform all steps of the protocol at room temperature unless otherwise stated. 1. Collect tissue and slice it to approximately 1–2 cm3 and rinse briefly in dilution buffer. 2. Immerse tissue immediately in 10% formalin. Volume of formalin should be at least 10–15 times the volume of the tissue. 3. Allow sample to rock slowly for 12–24 h at room temperature. 4. Discard fixative appropriately and wash 3
10 min with ample amount of dilution buffer by allowing samples to rock at room temperature. 5. Dehydrate the tissue and embed tissues in paraffin using the automated tissue embedder as described by others [6]. 6. Cut 3–10 μm thick sections using a microtome. Most commonly used thickness is 5 μm. 7. Collect tissue sections on positively charged slides (see Note 14). 8. Allow sections to air dry for 30 min at room temperature and then bake sections onto the slide using a slide warmer or oven at 40–45  C overnight or 60  C for 60 min. 9. Place the slides on a slide rack and deparaffinize the tissue sections by placing the slide rack in 2 changes of xylene (see Notes 3 and 24), 10 min each. 10. Hydrate the tissue sections by moving the slide rack into 2 changes of 100% ethanol for 5 min each, 95% and 70% ethanol for 3 min each. 11. Place the slide rack in distilled water to complete the hydration process. 3.2

Antigen Retrieval

3.2.1 Heat-Induced Epitope Retrieval

Tissue specimens that are fixed with formalin and subsequently embedded in paraffin require an antigen retrieval step prior to the IHC staining procedure. Formalin causes methylene bridges that cross-link the proteins and mask antigenic (epitope) sites. In order to break these bridges and expose the antigenic sites so that the antibody can bind, an antigen-retrieval step is required. This can be achieved by either heat-induced epitope retrieval (HIER) or protease-induced epitope retrieval (PIER) methods (see Note 5) [7]. 1. Place hydrated slides in a heat-proof slide container containing the appropriate HIER buffers and place in a microwaveable pressure cooker. Place two additional heat-proof slide containers filled with water on either side of the container for stability. 2. Fill a third of the pressure cooker with water and seal it closed. 3. Microwave the sealed pressure cooker for 10 min.

Immunochemistry on Tissue

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4. Wait for pressure to subside and carefully open it. Caution: The steam could be very hot. Use hot handle holder. 5. Place the container with the slides at room temperature and allow to cool for 20 min. 6. Rinse slides in deionized water. 3.2.2 Protease-Induced Epitope Retrieval

1. Place hydrated slides in a humidified slide staining chamber. 2. Cover sections with appropriate enzyme solution and incubate for 10–20 min at 37  C. 3. Allow sections to cool to room temperature for 10 min. 4. Rinse sections in water 2
3 min on a rocker/shaker.

3.3

Permeabilization

When the target antigen is intracellular, a permeabilization step using detergents is required. Permeabilization is also required for cytoplasmic epitopes of transmembrane proteins. Harsh detergents can disrupt the proteins, while mild detergents do not dissolve the plasma membrane (see Notes 15 and 24) [8]. 1. Working one slide at a time, wipe the backs of slide and gently dry the area around the slide using a fiber-free paper towel. 2. Draw a hydrophobic circle using a Dako pen around the slidemounted tissue and place flat in a humidified chamber (see Note 12). 3. Place a small drop of water over the section to prevent it from drying out, while allowing the Dako pen outline to air dry. 4. Repeat for all the slides. 5. For harsh permeabilization: Blot away the water from the slides by gently tapping the slide on a paper towel. Add sufficient detergent (100–200 μL) to cover the tissue sections and incubate at room temperature for 10 min. 6. For mild permeabilization: Blot away the water from the slides by gently tapping the slide on a paper towel. Add sufficient detergent (100–200 μL) to cover the tissue sections and incubate at room temperature for 30 min. 7. Place slides in a container with a slide rack filled with dilution buffer and rinse on a rocker for 5 min (see Note 24).

3.4

Blocking

In order to reduce nonspecific binding, this step is required [9]. Hydrogen peroxide suppresses endogenous peroxidase activity, thereby reducing background staining. This step is required only if the HRP label system is used. If using alkaline phosphatase substrate detection, omit the peroxidase quenching step and go directly to step 4 (see Notes 7, 24).

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1. Replace the container of dilution buffer with the aqueous peroxidase blocking solution (see Note 7). 2. Incubate for 10 min at room temperature. 3. Rinse in dilution buffer once for 5 min. 4. Lay the slides on the humidified chamber and add sufficient universal blocking buffer (100–200 μL) to cover the slides taking care not to let them dry. 5. Incubate at room temperature for 30 min. 6. Blot the universal blocking solution from the slides by gently tapping the slide on a paper towel. 7. If the avidin/biotin-based IHC protocol is being used, an additional avidin/biotin block is required (steps 8–11). 8. Apply avidin blocking solution for 15 min at room temperature. 9. Briefly rinse slide in dilution buffer. 10. Apply biotin blocking solution for 15 min at room temperature. 11. Briefly rinse the slide in dilution buffer to eliminate residual biotin. 3.5 Immunohistochemical Staining

Enzymatic IHC exploits the principle of antibodies binding specifically to antigens in biological tissues. The antibody is first labeled with the enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase (AP) before the reaction. After binding to the antigen, the labeled antigen forms an antigen-antibody complex where the enzyme catalyzes a specific substrate to yield an insoluble colored product. This insoluble product is then visualized using a light microscope. This approach can be performed using either direct or indirect detection methods [10]. The direct method is a one-step staining method that involves a primary antibody to be directly conjugated to a label (e.g., HRP-conjugated antibody). This labeled antibody is allowed to directly bind with the antigen of interest. The antigen-antibodylabel complex is then allowed to react with a substrate for staining. This method is suitable when the target antigen is abundantly present in the tissue or when the experiment calls for multicolor experiments. The indirect IHC detection method is frequently used for poorly expressed antigens and when signal amplification is required (see Note 24). The primary antibody that binds the antigen is then bound by a labeled secondary antibody raised against the host species of the primary antibody. Signal amplification can be achieved by the use of two or more labeled secondary antibodies. This however requires additional blocking steps and additional controls. For examples, when using avidin or streptavidin with

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biotinylated secondary antibodies to amplify the signal, an additional blocking step using a mixture of 0.001% avidin/0.001% streptavidin in dilution buffer is required. The amplification of the signal occurs when each avidin or streptavidin molecule binds four molecules of biotinylated secondary antibodies. Appropriate controls are of utmost importance for the accurate interpretation of IHC results. (see Note 16). 1. Blot the slides containing the blocking buffer by gently tapping the slides on a paper towel. 2. Apply 100–200 μL primary antibody diluted in universal blocking buffer (see Note 17). 3. Incubate overnight at 4  C in a humidified chamber (see Note 24). 4. Rinse 3
5 min in wash buffer with gentle agitation (see Note 18). 5. Apply 100–200 μL of enzyme (HRP or AP)-conjugated secondary antibody to the section and incubate for 30–60 min at room temperature. If using an HRP label for detection, incubate the slides in aqueous peroxidase blocking buffer (see Note 7). 6. Rinse 3
5 min in wash buffer with gentle agitation. 7. Apply 100–200 μL of DAB/AEC peroxidase substrate solution (for HRP-label) or New Fushcin alkaline phosphatase substrate solution (for AP-label) to cover the entire tissue section and allow color to develop (1–10 min) (see Note 19). 8. Monitor the development and intensity of the staining under a light microscope. As the enzyme catalyzes the chromogenic substrate, the insoluble colored product will localize to the sites of antigen expression. Compare the development of the color to the negative controls (see Notes 20 and 24). 9. Rinse in running tap water for 5 min. 10. Counterstain if required. To perform nuclear counterstaining, add enough drops of hematoxylin to cover the tissue section and incubate for 1 min (see Notes 11 and 21). 11. Rinse with warm tap water until color runs clear. 12. Incubate in warm tap water for 3 min. 13. Rinse 1 x 5 min in deionized water with gentle agitation. 14. Add mounting medium to cover the section and place a coverslip over the sections, taking care to avoid air bubbles (see Notes 2, 4, 11, 22, and 23). 15. Visualize staining of tissue under a bright field microscope (Fig. 2).

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Fig. 2 Representative image of a human lung section stained for the protein DSG2 using IHC. DAB was used for the detection of the label and the nuclei were counterstained with hematoxylin

4

Notes 1. For making all buffers, start with water in a measuring cylinder or a glass beaker with a magnetic bar. Place the container on a magnetic stir plate. Add the salts one at a time, mix using the stir bar and allow each salt to go into solution first before adding the subsequent salt. Warm the solution gently to 37  C if the salts take longer to go into solution. The solution should be at room temperature before adjusting the pH. 2. Some commercial formalin solutions contain methanol as an additive to prevent formaldehyde polymerization over time. Methanol could affect the way proteins are fixed in the tissue. Methanol causes proteins to precipitate, while formalin causes cross-linking of proteins. Additionally, formalin can degrade over time resulting in the production of formic acid. Methanol-free single-use formaldehyde ampules are now commercially available for safer and easier handling. Alternatively, the solution can be prepared fresh from crystalline paraformaldehyde. 3. Xylene is popular clearing agent and multiple changes are required to completely displace ethanol in the tissue. However, it is a toxic reagent. Less-toxic alternatives are preferred such as naphthenic solvents (e.g., Formula 83™), d-Limonene solvent (e.g., Hemo-De®), or xylene substitute. Xylene substitute does not tolerate water and when it becomes contaminated, the

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11

water droplets will form on the bottom. Additionally, when an excess of alcohol is carried over into xylene substitute during the dehydration process, the water present in the alcohol will cause it to become cloudy. 4. Toluene-based mounting medium is compatible when the tissue sections are cleared with xylene. When a xylene substitute is used, pay attention to compatible mountants (e.g., Thermo Scientific Shandon Xylene Substitute Mountant or equivalent). Xylene substitute is immiscible in anhydrous methyl alcohol and may not be compatible with several mounting medium products. 5. There is no general rule for the choice of antigen retrieval method used. Testing several antigen retrieval methods may be required to achieve optimal immunohistochemical reaction. HIER is the most commonly used method. De-masking of the epitope is more efficient at shorter treatment periods at higher temperatures and longer incubation times at lower temperatures. HEIR can be performed using a pressure cooker, a microwave, a vegetable steamer, or a water bath (overnight incubation set to 60  C). For most antigens, the optimal epitope recovery is obtained at a basic pH and EDTA buffer is particularly useful when working with over-fixed samples. PIER methods result in changes to tissue morphology and antigen, which makes it less popular. It is recommended to search the literature for what has been reported about the antigen/antibody, follow recommendations from antibody supplier if available, and start with HEIR method with a neutral pH during the optimization steps. Optimal incubation time for PIER may also vary and should be optimized. 6. Several tissues have endogenous peroxidase activity. If HRP label is being used for IHC, then it is advisable to determine the endogenous peroxidase levels by reacting the fixed tissue sections with DAB substrate. If brown staining appears, then the endogenous peroxidase activity can be quenched using peroxide blocking solution. Many tissues such as kidney, intestine, lymphoid tissue, and placenta contain high content of endogenous alkaline phosphatase. Hence, if AP label is being used, it is advisable to first determine the endogenous AP levels by reacting the tissue with BCIP/NBT (e.g., 1-Step™ NBT/BCIP substrate solution, ThermoFisher) or New Fuschin. If a blue or red color develops, respectively, then the endogenous AP needs to be blocked using levamisole which is added to the chromogenic substrate. 7. Blocking of endogenous peroxidase can be performed either before the universal blocking step or after the primary incubation step. Some epitopes are modified by peroxide and in this

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case, quenching can be performed after the primary antibody incubation step. The peroxide solution can be prepared in methanol, PBS, or water and the nature of the tissue determines the choice. Blood smears and tissues rich in peroxidase can be damaged in aqueous peroxide solution. In this case, peroxide in methanol is preferred. Certain cell surface antigens are sensitive to methanol, and in this case, peroxide in either PBS or water is recommended. 8. The universal blocking buffer can be used to dilute the unconjugated primary antibody. This buffer cannot be used to dilute HRP- or AP-conjugated antibodies since the sodium azide in the recipe is an inhibitor of HRP and the phosphate in the dilution buffer (if PBS is being used) is an inhibitor of AP. 9. Some tissues contain endogenous biotin, such as liver and kidney. When the biotin-avidin detection system is used, it will be necessary to eliminate the binding of endogenous biotin to avidin using the avidin/biotin blocking step. Here, the tissue is first pre-treated with unconjugated avidin, and then saturated with biotin. 10. DAB and AEC substrates are used when HRP is used as the label. The pH of the DAB substrate is crucial. A pH less than 7.0 will cause decrease in staining intensity and a pH above 7.6 will cause significant background staining. 11. AEC-stain and New Fuchsin stains are ethanol soluble and hence alcohol-containing solutions for counter-staining (e.g., Harris’ hematoxylin, acid alcohol) or alcohol/toluene-based mounting medium should not be used. The slides must be coverslipped using aqueous mounting medium. 12. An easy and inexpensive way to make a humid chamber would be to use a shallow plastic container with a lid. Place a layer of wet paper towels at the bottom. Align two cut serological pipettes and place them in the container with parallel orientation, about 2 inches apart. Place a single sheet of paper towel over the pipettes and spray it with water to wet it. This will provide traction. The slides can be placed to lie flat on top of the pipettes during the incubation steps. 13. The appropriate time needed for fixation depends on the size of the tissue and type of tissue. It is important that the tissue is completely submerged in sufficient excess volume of fixative. Ideally 18–24 h of incubation time is sufficient for most tissues. Extremely fibrotic tissue that is over 0.5 cubic inch may need longer incubation times. Under-fixation of the tissue will lead to strong staining in the edge of the tissue, with little to none staining in the center of the tissue. Over-fixation will cause issues with masking of the epitopes that may or may not be unmasked with antigen retrieval.

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14. Sometimes the tissue sections are not completely flat or the slides are not completely clean. Additionally, the IHC protocol involves several harsh steps and the risk of losing the tissue from the glass slide is high. In order to avoid this, adhesive-coated slides (such as silane or poly-L-lysine) or positively charged slides are required. 15. The time and concentration of the detergent used depends on the localization of the antigen. Harsh detergents are highly suitable for nuclear antigens (e.g., 0.2% harsh detergent for 10 min). For cytoplasmic antigens, the concentration of the harsh antigen and the time of treatment should be reduced (e.g., 0.1% harsh detergent for 5 min). Sometimes organic solvents such as methanol or acetone can be used to fix tissue. These solvents remove lipids and dehydrate the cells and cause precipitation of the proteins. No permeabilization step is needed when organic solvents are used to fix the tissue. 16. Controls to be included. (a) A no-antigen retrieval control slide to determine artifactual staining. (b) Positive control using a tissue known to express an antigen and that has an IHC protocol with an antibody previously established. This helps to make sure that the protocol works. (c) Negative control to test the specificity of the antibody. Ideally, the tissue section for the negative control should be on the same slide as the test section to eliminate any time-related inconsistencies. For the negative control, omit the primary antibody, and instead incubate the section with the universal blocking buffer or with normal serum from the same species as the primary antibody. Additional controls to support the specificity of staining can include absorption controls and tissue type controls. 17. Start with the primary and secondary antibody concentrations recommended in the datasheet. If the primary antibody concentration it is not available, then a series of antibody concentrations ranging from 0.5 to 5 μg/mL is recommended. For unpurified antibodies, testing a range of concentrations starting from undiluted (neat) to 1:100 can be tested. 18. It is crucial here to use a separate wash container for each kind of primary antibody used to avoid cross-contamination. Also, if the test tissue and the negative control tissue are on the same slide, care should be taken to avoid reagent from the test tissue contaminating the negative control tissue. This can be avoided by either holding the slide down vertically over a waste container such that the test section in below the negative control section. Then using a plastic pasteur pipette, rinse the sections

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from the top closer to the negative control section so that concentrated antibody is first rinsed off into the waste container before returning the slide into the wash container for further rinsing. 19. Signal amplification can be used for detection of low expressing antigens or to improve results from poorly performing antibodies. Labeled streptavidin-biotin (LSAB) method or tyramide signal enhancing (TSE) can be used. For the LSAB method, a biotinylated antibody against the host the primary antibody is applied to the tissue after the primary incubation step. This is followed by incubation with a high sensitivity Streptavidin-HRP conjugate, which can then be detected using a chromogen for HRP (DAB or AEC). In TSE, the peroxidase-catalyzed reaction is subsequently made to covalently attach a tyramide portion of tyramine-protein conjugates close to the antigen of interest after the primary- and secondary-HRP conjugate incubation steps. An antibodyenzyme or a fluorophore conjugate is then made to react to the protein-portion of the tyramine-protein conjugate which can be detected. 20. DAB and AEC are hazardous materials. Appropriate PPE should be worn and all steps should be performed in a chemical fume hood. Follow MSDS for proper disposal. 21. Hematoxylin counterstain can make visualization of targets localized in cell nuclei difficult. 22. Blot excess water or solvent from the non-sample surface of slide. Apply a small amount (approximately 50 μL) of mounting medium to the surface of the slide and slowly tip a coverslip onto the mounting medium, gently lowering it into place and avoid creating bubbles. Allow the mounting medium to cure per the manufacturer’s instruction. Seal the sides of the coverslip with nail polish if required. 23. If DAB reagent is being used, the slides can be dehydrated by incubating them with gradual increase in ethanol concentration (70%, 90%, 100%) and a final 2
change of xylene for 5 min each. Make sure to blot excess solvent as much as possible before making the changes into the solvent tubs to prevent carryover of the solvents. These slides can then be mounted using a toluene-based mounting medium for longterm storage. AEC and New Fuschin are soluble in alcohols and xylene. Hence, when these chromogens are used, the slides should be coverslipped using only aqueous mounting media. 24. For troubleshooting on staining intensity, specificity, and background signal, please see Table 1.

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Table 1 Troubleshooting guide for immunohistochemistry Problem

Reason

No staining

Antigen is not present in the tissue

Use a positive control recommended in the datasheet l Antigen is not abundant Use signal amplification step l Increase concentration of primary antibody l Increase incubation time of primary antibody l Use harsh detergents or increase incubation Improper permeabilization time l Over-fixation of samples Standardize antigen retrieval l Fix tissue for lesser time Excess wax in tissue Clear tissue in xylene longer Antibody is unsuitable for IHC Test antibody in non-denatured immunoblot to make sure it is not damaged Primary and secondary antibodies are Make sure that that secondary antibody is not compatible against the species that the primary antibody was raised in Antibodies or reagents are damaged Run positive control

Tissue sections allowed to dry out High background High primary antibody concentration staining High incubation temperature Excess wax in tissue Endogenous enzyme activity blocking was insufficient Inadequate protein blocking Inadequate washing steps Too much substrate Amplification step not needed Unspecific staining

Solution

Antibody concentrations are too high Endogenous enzyme activity blocking was insufficient The primary antibody is raised in the same species as the tissue being stained Tissue sections allowed to dry out

Use humidified chamber Titrate the antibody concentration Incubate at 4  C Clear tissue in xylene longer Increase incubation time or concentration Increase incubation time with universal blocking solution Increasing washing steps and times Reduce substrate incubation time. Remove amplification step Reduce and titrate antibody concentrations Increase incubation time or concentration Use a primary antibody raised in a different species to that of the tissue being stained Use humidified chamber

References 1. Nakane PK, Pierce GB Jr (1966) Enzymelabeled antibodies: preparation and application for the localization of antigens. J Histochem Cytochem 14:929–931 2. Mason DY, Sammons R (1978) Alkaline phosphatase and peroxidase for double immunoenzymatic labelling of cellular constituents. J Clin Pathol 31:454–460

3. Faulk WP, Taylor GM (1971) An immunocolloid method for the electron microscope. Immunochemistry 8:1081–1083 4. Gedda L, Bjorkelund H, Andersson K (2010) Real-time immunohistochemistry analysis of embedded tissue. Appl Radiat Isot 68:2372–2376 5. Werner M, Chott A, Fabiano A, Battifora H (2000) Effect of formalin tissue fixation and

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processing on immunohistochemistry. Am J Surg Pathol 24:1016–1019 6. Hewitson TD, Wigg B, Becker GJ (2010) Tissue preparation for histochemistry: fixation, embedding, and antigen retrieval for light microscopy. Methods Mol Biol 611:3–18 7. MacIntyre N (2001) Unmasking antigens for immunohistochemistry. Br J Biomed Sci 58:190–196

8. Jamur MC, Oliver C (2010) Permeabilization of cell membranes. Methods Mol Biol 588:63–66 9. Miller RT (2019) Avoiding pitfalls in diagnostic immunohistochemistry-important technical aspects that every pathologist should know. Semin Diagn Pathol 36:312–335 10. Ramos-Vara JA (2017) Principles and methods of immunohistochemistry. Methods Mol Biol 1641:115–128

Chapter 2 Indirect Immunofluorescence of Tissue Sections Cody J. Aros Abstract Immunofluorescence (IF) on tissue sections allows for the detection of protein species subcellular localization. IF studies further offer the ability to achieve this understanding at the level of single cell granularity. Here, we describe the processes by which tissue is fixed, embedded, sectioned, and subsequently utilized for conducting indirect IF assays. We raise potential opportunities for troubleshooting and optimization at varying stages of the protocol. Key words Immunofluorescence, Antibody, Antigen retrieval, Permeabilization, Blocking

1

Introduction Immunofluorescence (IF) involves the detection and visualization of protein targets on slides of sectioned tissue. Broadly, IF can be divided into two forms: direct and indirect. Direct IF entails the use of a primary antibody that is conjugated to a visualization label, such as a fluorophore [1, 2]. Indirect IF utilizes two distinct incubation steps: first, a primary antibody incubation followed by a secondary antibody incubation step that uses an antibody that targets antigens of the host in which the primary antibody was raised [3]. Fixation is the first step in conducting IFs, which preserves the cytoarchitecture of the cell to render them poised for target detection. Following tissue embedding in paraffin and sectioning, slides must undergo a series of washes to de-paraffinize the tissue. Next, antigen retrieval is performed to expose the epitope targets across the tissue. If tissue is embedded in optimal cutting temperature (OCT) compound, de-paraffinization and antigen retrieval steps are not necessary. Next, permeabilization allows for antibody accessibility to the target of interest. Tissue blocking minimizes background IF staining by binding to nonspecific epitopes that would otherwise be detected by the primary antibody. Subsequent

Aik T. Ooi (ed.), Single-Cell Protein Analysis: Methods and Protocols, Methods in Molecular Biology, vol. 2386, https://doi.org/10.1007/978-1-0716-1771-7_2, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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primary and secondary antibody incubations allow for detection and visualization of the targets [3]. Here, we describe each of these steps of protocol in detail with accompanied optimization points that can be employed to generate reliable detection of the target of interest.

2

Materials Prepare all solutions using de-ionized water and analytical grade reagents. All buffers should be prepared and stored at room temperature unless otherwise indicated. Hazardous and toxic reagents should be properly disposed.

2.1

Tissue Fixation

1. 10X Tris-Buffered Saline (TBS) stock, pH 7.6: Add 700 mL de-ionized water to 1 L graduated cylinder. Place magnetic stir bar in graduated cylinder. Place graduated cylinder on magnetic platform and turn on to rotate stir bar. Weigh 24.2 g Tris base and 80 g NaCl and add each to the graduated cylinder. Adjust pH with 5 N HCl to 7.6. Bring final volume to 1 L using de-ionized water. 2. 1X TBS with 0.05% Tween-20 (TBST): Dilute 100 mL 10X TBS stock in 900 mL de-ionized water. Add 500 μL Tween-20 to solution and invert to mix. 3. Tissue fixative: 4% paraformaldehyde (PFA). Under a chemical fume hood, dilute 10 mL of 16% formaldehyde in 30 mL of 1X TBST. Store at 4
C (see Note 1). 4. 10X Phosphate-Buffered Saline (PBS) stock, pH 7.4: Add 700 mL de-ionized water to 1 L graduated cylinder. Place magnetic stir bar in graduated cylinder. Place graduated cylinder on magnetic platform and turn on to rotate stir bar. Weigh 4.56 g NaH2PO4, 11.5 g Na2HPO4, and 87.68 g NaCl (see Note 2). Adjust pH with 5 N HCl to 7.4. Bring final volume to 1 L using de-ionized water. 5. 1X PBS: Dilute 100 mL 10X PBS stock in 900 mL de-ionized water. 6. 20% sucrose: Weigh 20 g sucrose and place in glass beaker with magnetic stir bar. Add 100 mL 1X PBS. Mix until dissolved. Store at 4
C (see Note 3).

2.2 Tissue Deparaffinization

1. Slides with cross-section of tissue 2. Heating block 3. Slide containers 4. Xylene (see Note 4) 5. 100% Ethanol

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6. 90% Ethanol 7. 70% Ethanol 8. De-ionized water 2.3

Antigen Retrieval

1. 1 mM EDTA, pH 8.0. (see Note 5). 2. Microwave pressure cooker 3. Heat-proof slide containers 4. De-ionized water 5. Microwave

2.4 Tissue Permeabilization

1. Kimwipes or similar cleaning wipes 2. Hydrophobic PAP Pen 3. Wet paper towels 4. Slide box (serves as humidified chamber with paper towels) 5. 1X TBST 6. Coplin jar 7. Permeabilization buffer: 0.5% Triton X-100 in 1X TBST (see Note 6). Add 50 μL of Triton X-100 to 10 mL 1X TBST. Vortex to allow Triton X-100 to become miscible.

2.5 Tissue Blocking and Antibody Incubations

1. Dako Blocking Buffer (see Note 7). Store at 4
C 2. Primary antibodies 3. Fluorophore-conjugated secondary antibodies 4. 5 mg/mL 40 ,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI)

2.6

Slide Mounting

1. Vectashield Mounting Media (see Note 8) 2. Coverslips 3. Nail polish 4. Slide box

3

Methods Conduct all steps of the protocol at room temperature unless otherwise indicated. An overview of the workflow is depicted in Fig. 1.

3.1

Tissue Fixation

1. Place tissue of interest in 4% PFA solution. Allow sample to rock slowly at room temperature (see Note 9). 2. On the following day, remove samples from rocker. Under chemical fume hood, remove PFA by decanting or pipetting

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Fig. 1 Schematic indicating expected timeline for each step of the protocol, indicated in blue: tissue fixation, tissue de-paraffinization, antigen retrieval, permeabilization, tissue blocking, primary antibody incubation, secondary antibody incubation, and mounting. Paraffin embedding and tissue sectioning, indicated in yellow, can have variable durations in time

into appropriate waste bottle. Add approximately 10X the volume of tissue of 1X PBS to each sample and allow samples to rock at room temperature for 10 min. 3. Remove PBS by decanting or pipetting into appropriate waste bottle. Conduct an additional 2 washes with 1X PBS, rocking at room temperature for 10 min. 4. If embedding tissue in OCT, add 10X the volume of the tissue of 20% sucrose to each sample. Allow sample to rock slowly overnight at 4
C (see Note 10). 5. For OCT and paraffin-embedding of tissue sample, perform as described by others [4]. 3.2 Tissue Deparaffinization

This section is only completed if tissue was embedded in paraffin. 1. Melt paraffin from tissue slides and bake sections onto slides for proper adherence by placing slides on heating block set to 60
C for 1–2 h (see Note 11). 2. Perform dewaxing and hydration of tissue sections on slides with the following incubations in slide containers under a chemical fume hood: xylene for 15 min, xylene for 15 min, 100% ethanol for 5 min, 100% ethanol for 5 min, 90% ethanol for 5 min, 90% ethanol for 5 min, 70% ethanol for 5 min, and de-ionized water for 5 min.

3.3

Antigen Retrieval

This section is only completed if tissue was embedded in paraffin. 1. Place de-paraffinized slides in a heat-proof slide container containing 1 mM EDTA, pH 8.0. Perform a heat-based antigen retrieval by first placing the container with slides in the pressure cooker (see Note 12). Place two additional heat-proof containers filled with de-ionized water on either side of the container with slides. Fill pressure cooker with water and seal it closed as depicted in Fig. 2.

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21

Fig. 2 (a) Image of open pressure cooker filled with water and three heat-proof slide containers. (b) Image of sealed, closed pressure cooker prior to placement in microwave for antigen retrieval

Fig. 3 Image of tissue sections with outlined boundaries drawn by hydrophobic PAP pen

2. Microwave the sealed pressure cooker for 10 min. 3. Carefully open pressure cooker and place slides with container of EDTA on ice for 10 min. Allow to cool. 3.4

Permeabilization

1. Remove slides from EDTA and dry backs of slides and area around tissue with Kimwipes. 2. Outline sections of tissue by drawing boundaries with hydrophobic PAP pen. Allow PAP pen outlines to air dry as shown in Fig. 3. 3. Place two wet paper towels on the inside of a slide box to create a humidified chamber.

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4. Place slides lying flat in humidified chamber. 5. Prepare fresh 0.25–0.5% Triton X-100 diluted in 1X TBST. Permeabilize tissue by pipetting enough of the Triton X-100 solution onto each section to cover the entirety of the tissue. Make sure not to allow sections to dry. Close the humidified chamber and incubate for 10 min. 6. Remove Triton X-100 solution and place slides in coplin jar filled with 1X TBST. Wash slides on rocker for 5 min. 3.5 Tissue Blocking and Antibody Incubations

1. Remove slides from coplin jar of 1X TBST and lie each of them flat in humidified chamber. 2. Block tissue by pipetting enough of the Dako Blocking Buffer onto each section to cover the entirety of the tissue. Close the humidified chamber and incubate for 1 h (see Note 13). 3. Decant Dako Blocking Buffer off of tissue sections and place back in humidified chamber. 4. Dilute primary antibodies in Dako Blocking Buffer. Pipet enough of primary antibody solution onto each section to cover the entirety of the tissue. Close the humidified chamber. 5. Incubate overnight at 4
C (see Note 14). 6. Decant primary antibody solution off of tissue sections. 7. Fill coplin jar with 1X TBST and place slides in it. Incubate on rocker at room temperature for 5 min. 8. Remove slides from coplin jar and decant 1X TBST. Replace with fresh 1X TBST and place slides back in coplin jar. Incubate on rocker at room temperature for 5 min. Repeat 1X TBST wash once more. 9. Place slides facing upright in humidified chamber. Dilute secondary antibodies and DAPI in Dako Blocking Buffer. Pipet enough of secondary antibody solution onto each section to cover the entirety of the tissue. Close humidified chamber. 10. Incubate slides in secondary antibody solution for 1 h at room temperature in darkness (see Note 15). 11. Decant secondary antibody solution off of tissue sections. 12. Fill coplin jar with 1X TBST and place slides in it. Incubate on rocker at room temperature for 5 min in darkness. 13. Remove slides from coplin jar and decant 1X TBST. Replace with fresh 1X TBST and place slides back in coplin jar. Incubate on rocker at room temperature for 5 min in darkness. Repeat 1X TBST wash once more. 14. Remove slides from coplin jar and decant 1X TBST. Replace with fresh 1X TBS and place slides back in coplin jar. Incubate on rocker at room temperature for 5 min in darkness.

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23

15. Remove slides from coplin jar and decant 1X TBS. Replace with fresh de-ionized water and place slides back in coplin jar. Incubate on rocker at room temperature for 5 min in darkness. 3.6

Slide Mounting

1. Remove slides from coplin jar and decant de-ionized water. Dry the back side of each tissue slide using a Kimwipes. Dry the regions of the front of the slide that does not contain tissue. Allow the slides to air-dry in a slide box in darkness. 2. Pipet a small amount of Vectashield anti-fade mounting media to air-dried tissue sections, enough to cover the surface of around 25% of the tissue. 3. Place one end of the coverslip on the slide. Using forceps, gently lower the opposite side of the coverslip onto the slide, avoiding bubbles. 4. Begin sealing the mounted sections by placing a small drop of nail polish on each of the four corners of the slide. Allow to air dry for approximately 5–10 min in darkness. 5. Seal the remaining portions of the slide by applying nail polish along the edge of the coverslip and tissue slide. Allow to air dry for 10 min in darkness. 6. Store in 4
C in a slide box in darkness until ready for imaging.

4

Notes 1. Formaldehyde should be exclusively handled under the chemical fume hood, as it can cause toxicities upon inhalation or ingestion. It should furthermore be disposed of in an appropriate disposal container in adherence with oversight environmental health and safety protocols. 2. Collectively, these powdered reagents take several minutes to dissolve into solution. 3. 20% sucrose solution is prepared fresh each instance the protocol is conducted to minimize possibility of contamination. 4. Xylenes are an important reagent to facilitate de-paraffinization. However, xylenes are known to cause adverse health effects upon inhalation or ingestion. As such, it is important to work under a chemical fume hood when working with xylenes. Other groups have reported using HistoClear in lieu of xylenes. We find, however, that use xylenes yield improved immunofluorescence staining intensity in comparison that observed when Histo-Clear is used. 5. We have used both powdered EDTA dissolved in de-ionized water or purchased EDTA from commercially available sources.

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Both yield similar efficacies of immunofluorescence staining. Other antigen retrieval buffers may also be considered such as those that use citrate or trypsin. 6. The permeabilization step is important to allow for reliable detection of intracellular proteins. The percentage of Triton X-100 used in the permeabilization solution can be adjusted as deemed necessary. Low concentrations of Triton X-100 will yield less tissue permeabilization than that of higher concentrations. If the desired immunofluorescence stain is membranous, for example, no permeabilization is required. In contrast, if the desired target is nuclear, around 0.25–0.5% Triton X-100 should be used (Fig. 4). Further, other reagents can be used to accomplish tissue permeabilization if Triton X-100 is not desired. Use of alcohol-based products such as ethanol or methanol combine both fixation and permeabilization and can therefore improve target signal detection.

Fig. 4 Representative IF images of normal human airway epithelium (left) and squamous lung cancer sample (right) stained using 0.5% Triton X-100 permeabilization to allow for detection of nuclear p-β-cateninY489 (red) accumulation. DAPI (blue) indicates the nucleus and K5 (green) indicates Keratin 5, the marker of the adult airway basal stem cell

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7. Other block reagents can be used as an alternative to Dako Blocking Buffer. These options include normal serum, milk, or bovine serum albumin to reduce nonspecific binding of the primary antibody. 8. The Vectashield mounting media can be used with or without DAPI. If using without DAPI and are interested in detecting the nucleus, DAPI (0.1–1 μg/mL final concentration) can be incorporated in the secondary antibody incubation. 9. The amount of time the tissue should be incubated in fixative is dependent on the size of the tissue. For example, mouse embryos should be fixed for 1–2 h, while larger tissues such as lung or brain should be fixed overnight. 10. Sucrose incubation of the fixed tissue is important, as it serves as a cryoprotectant for those that will be embedded in OCT. As such, it prevents the formation of ice crystals that would otherwise damage cell membranes. For larger tissues, it is important to do a series of sucrose gradient incubations. For example, incubate the tissue in 15% sucrose overnight followed by 25% sucrose overnight. 11. Use of positively charged slides will allow for better adherence of the tissue to the slide to uphold integrity throughout the protocol. 12. Antigen retrieval is usually not needed if the tissues were embedded in OCT. Other antigen retrieval methods can be considered if heat-based methods are not preferred in order to maximize target signal detection. For example, trypsin, pepsin, or proteinase K solutions can be used to conduct enzymatic antigen retrieval. However, these reagents may result in compromised tissue morphology. Heat-based antigen retrieval is useful for tissue for which morphology must be maintained. If working with tissue that slides off the slide upon high pressure incubations, slides can alternatively be incubated in a 60
C water bath overnight. 13. If looking for a place to temporarily stop the protocol, the blocking incubation can alternatively incubate overnight at 4
C. 14. If desired, some primary antibodies can be incubated on tissue sections for 1 h at room temperature and yield comparable target signal intensity. 15. Secondaries antibody incubations and subsequent sample handling from this step should be conducted in darkness. Exposure of samples to light at this point may result in photobleaching of the fluorophores, thereby compromising the ability to detect adequate target signal.

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References 1. Odell ID, Cook D (2013) Immunofluorescence techniques. J Invest Dermatol 133(1):1–4 2. Aoki V, Sousa JX, Fukumori LMI, Pe´rigo AM, Freitas EL, Oliveira ZNP (2010) Direct and indirect immunofluorescence. An Bras Dermatol 85(4):490–500 3. Im K, Mareninov S, Diaz MFP, Yong WH (2019) An introduction to performing

immunofluorescence staining. Methods Mol Biol 1897:299–311 4. Hewitson TD, Wigg B, Becker GJ (2010) Tissue preparation for histochemistry: fixation, embedding, and antigen retrieval for light microscopy. Methods Mol Biol 611:3–18

Chapter 3 Flow Cytometry for Beginners: Hints and Tips for Approaching the Very First Single-Cell Technique Valentina Proserpio, Laura Conti, and Salvatore Oliviero Abstract While many single-cell proteomics techniques have been rapidly developed over the past decade, flow cytometry still remains the pillar of single-cell protein analysis, as it allows to rapidly analyze and characterize protein expression in millions of cells. In this chapter, we will describe the main steps to prepare and acquire samples for flow cytometry, with particular focus on the setup of the right controls that are instrumental in analyzing and interpreting the results. Key words Single cell, Protein quantification, Flow cytometry

1

Introduction In the last decade, the almost uncontrolled development of several biomolecular techniques, the so-called single-cell Omics, have allowed scientists to analyze the transcriptomic, the genomic, and also the proteomic profile of individual cells. Nevertheless, the very first single-cell technique that has been developed more than 50 years ago is the Flow Cytometry (FC) [1]. FC is a laser-based technology for measuring fluorescence intensity emitted by either fluorescent-labeled antibodies against specific proteins or fluorescent molecules that are bound to cells or cell components like nucleic acids or ligands. In a classical FC experiment, the sample is represented by individual cells labeled with fluorophores-conjugated antibodies, resuspended in solution and then run through a flow cytometer in a narrow stream where a laser beam will hit one cell at a time (see Fig. 1).

Valentina Proserpio and Laura Conti contributed equally with all other contributors. Aik T. Ooi (ed.), Single-Cell Protein Analysis: Methods and Protocols, Methods in Molecular Biology, vol. 2386, https://doi.org/10.1007/978-1-0716-1771-7_3, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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Fig. 1 Schematic diagram of the flow cytometer. The light emitted by the lasers hits the cells as they flow one by one. After hitting a cell, the light will be scattered in all the directions and different detectors will capture the signal that will be finally analyzed and then processed in silico

When cells are hit by the beam, the light scatter is detected. The detector in front of the laser beam will detect the “Forward Scatter or FSC” that will be roughly proportional to the size of the cells, while a detector on the side of the light beam will detect the “Side Scatter or SSC” that represents a proxy of the granularity of the cells. Dye-specific fluorescence detectors will be responsible for acquiring the information of different fluorescent molecules. The data collected by all the detectors will be visualized in classical two-dimensional dot plots in which each dot represents one event (or one cell in our case) -or in density or contour plotsand the two main axes will be the amplitude of the signal acquired. Axis can be in either linear (for FSC and SSC) or logarithmic or biexponential scale (for SSC and fluorescent signals). Just by FSC and SSC different cells in suspension can be divided in groups based on their relative sizes and level of granularity. One easy example can be represented by total blood samples as in Fig. 2. For the analysis of leukocyte populations in blood or organs, panels including antibodies directed to the markers of the main populations are used. For example (Fig. 3), to analyze T and NK cells in mouse spleen, a first dot plot with FSC vs SSC can be used to easily distinguish splenocytes from debris while a second plot of FSC-A (area) versus FSC-H (height) can distinguish singlets from duplets. 7-AAD-negative cells represent live cells and NK cells are then recognized by being CD45 and CD49b positive. T cells are identified as CD3 positive and either CD4 or CD8 positive for CD4 T helper cells or CD8 T cytotoxic cells respectively. In this chapter, we will illustrate the main steps required for basic flow cytometry experiments.

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250K Granulocytes

SSC-A

200K

150K

100K Lymphocytes

Monocytes

50K

0 0

50K

100K 150K FSC-A

200K

250K

Fig. 2 Example of total blood FSC-A versus SSC-A plot. The three main populations of lymphocytes, monocytes, and granulocytes can be distinguished based on relative size and granularity

2 2.1

Materials Cell Preparation

1. PBS 2. BSA 3. Trypsin 4. Any medium with FBS 5. Collagenases 6. EDTA 7. Red Blood Cells (RBC) Lysis Buffer (optional) 8. Filters (70/100μm) 9. Syringes 10. Scalpels 11. FACS tubes

2.2 Selection of the Best Panel

1. Table of spectrum/compatibility 2. Spectral websites that display excitation and emission curves for fluorochromes. Here some examples: – http://www.bdbiosciences.com/research/multicolor/spec trum_viewer/index.jsp – http://www.lifetechnologies.com/order/spectra-viewer – http://eu.ebioscience.com/resources/fluorplanspectraviewer – http://www.biolegend.com/spectraanalyzer

Fig. 3 Representative gating strategy illustrating T cell subsets and CD4+ T cell activation in BALB/c mouse splenocytes. Artifact exclusion by time gating, followed by FSC vs SSC to discriminate splenocytes, aggregate exclusion, and 7-AAD gating for single live cells. Leukocytes were gated as CD45+, the CD49b was used as an NK marker and CD3 as a T cell maker. CD4+ and CD8+ T cell subsets were identified, and CD69+ activated CD4+ T cells were gated based on FMO control (not shown)

30 Valentina Proserpio et al.

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Table 2 List of available viability dyes

Stain

Excitation/ Emission

Detection

Helix NP NIR

Dead cells only

640/660

Alexa 647/APC

Draq5

Dead cells only

488

695 LP

DRAQ7

Dead cells only

633/695

Alexa Fluor700/APC/ Cy7

7-AAD (7-aminoactinomycin D)

Dead cells only

488

650

Propidium Iodide (PI)

Dead cells only

488

PE/Texas Red

TO-PRO-3

Dead cells only

642/661

Live/Dead fixable dyes

Dead cells more than live Several colors cells available

Zoombie dyes

Dead cells more than live Several colors cells available

2.3

Staining of Cells

1. Ice cold PBS 2. Cell Counter or Bu¨rker chambers 3. Fc blocking antibody 4. Aluminium foil 5. Fixation Buffer: PBS 4% paraformaldehyde 6. Permeabilization Buffer: PBS 0.5% BSA 0.1% saponin 7. Wash buffer: PBS 0.5% BSA 8. Antibodies 9. Live staining dye as 7-AAD, TO-PRO-3, TOTO-3, propidium iodide, Hoechst 33258, and DRAQ5 (see Note 1 and Table 2 for details).

2.4 Facs Analyzer Setup

1. Calibration beads, needed to check the daily performances of the flow cytometer. The most used are Cytometer Setup and Tracking (CS&T) beads (BD Biosciences) and 8-peak Rainbow bead calibration particles (Spherotech). 2. Compensation beads, needed to perform compensation between fluorochromes in a multiparametric staining.

2.5 Data Acquisition and Data Analysis

1. Flow-Jo or similar softwares.

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Methods Cell Preparation

3.1.1 For Adherent Cells

In order to perform a good staining, it is important that cells are in a single-cell suspension without clumps. The number of cells per sample must be the same in all the samples. To do so, different samples may require different enzymes and different strategies, so we will list the most common. 1. Wash once with PBS. 2. Add any of the following directly to the cells. Trypsin is the most widely used enzymatic solution (see Note 2), but also Accutase, or Versene (0.5 mM EDTA in PBS) will do. 3. Block the reaction by adding complete medium. 4. Gently pipette the cells up and down several times. 5. Centrifuge and resuspend in the appropriate amount of PBS (see Note 3). 6. Count the cells with burker chambers or similar. See Note 4.

3.1.2 For Cells in Suspension

1. (Recommended) Add 0.2 mM EDTA in the PBS when resuspending the cells to avoid cells clumping (see Note 2). 2. (Recommended) Use viability dyes for these samples as it is more difficult to remove dead cells from cells in suspension (as compared to adherent cells). 3. Count the cells with burker chambers or similar devices. See Note 4.

3.1.3 For Tissues

After tissue resection, we recommend to keep the sample on ice all the time to preserve cell viability. Before performing enzymatic digestion, it is important to mechanically dissociate the tissue with a scalpel [2]. 1. Place a 10 cm petri dish on ice (you can also use the lid of the petri dish). 2. Place the tissue in the center and, with a clean scalpel, cut the tissue in all the directions several times. A properly digested tissue will look like a small meatball. 3. Collect the tissue with the scalpel and transfer it to a falcon tube. 4. Dilute collagenase at 1 mg/mL in PBS. Use up to 1 mL of collagenase per sample. 5. Cut the tip of a p1000 tip and resuspend the cells in the collagenase solution pipetting the tissue up and down several times until you reach a semi-homogenous solution. 6. Let the collagenase to work for 15 min on ice (see Note 5).

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7. Place a 70 or 100 μm strainer on top of a clean 50 mL falcon tube and transfer the solution. 8. Use the pressure from the pipette to filter the solution through the strainer. Collect most of the cells using the back of a syringe plunger to smash the tissue onto the strainer and use PBS to wash it down. 9. Centrifuge at 700
g for 4 min at 4  C. 10. If not interested in red blood cells, perform red blood cell lysis (optional) resuspending the cell pellet in RBC lysis buffer. 11. Wait for 5 min and block the reaction with 5 volumes of PBS (according to manufacturer’s instructions). 12. Centrifuge the cells at 700
g for 4 min at 4  C. 13. Resuspend the cells in PBS. 14. Count the cells with burker chambers or similar. See Note 4. 3.2 Selection of the Best Antibody Panel

In the design of a multiparametric panel for the simultaneous detection of different antigens, several characteristics of the flow cytometer and of the antigens need to be considered. In general, a variety of fluorochromes can be used, among which the most common are FITC, PE, PE-Cy5, PerCP-Cy5.5, PE-Cy7, APC, APC-Cy7, BV421, and BV510. 1. Check the configuration of your flow cytometer (refer to your instrument’s manual) and choose fluorochromes whose excitation wavelength is compatible with its lasers and whose emission wavelength matches the available filters and detectors. 2. Select fluorochromes with minimal spectral overlap, in order to minimize the need for compensation. To this end, check the excitation and emission curves of your candidate fluorochromes on the spectral websites indicated in the Material section. See Note 6. 3. Assign each marker a fluorochrome. To do this, you should consider the fluorochrome brightness and the cellular expression level of the antigens you are going to analyze. The simplest rule to consider is to use “the strongest fluorochrome to the weakest antigen.” Bright fluorochromes (such as PE or APC) should be conjugated to antibodies detecting low expressed antigens, while highly expressed antigens provide a good separation between negative and positive cell populations, so can be detected with dimmer fluorochromes (such as FITC or PerCP). See Note 7. 4. Perform antibody titration to determine which concentration has minimal background staining without significant loss of specific signal. See Note 8.

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5. Check antibody specificity. When using an antibody for the first time, especially if it is not largely in the literature, it would be a good practice to stain cells that are negative and cells that are surely positive for the antigen, to verify it works properly. 3.3 Controls Preparation

Flow cytometry experiments require the use of specific controls to allow for correct sample acquisition and data interpretation. The following control samples should be prepared and used in each experiment. 1. Autofluorescence control. Cells contain several molecules that absorb light at different wavelengths and emit signals that can be measured by the detectors. Acquisition of unstained cells is useful to determine this background signal, which usually increases with cell size. 2. Compensation controls. Use single color-stained samples, containing a positively stained population and a negative or unstained population. When possible, use BD CompBeads or polystyrene microparticles coupled to an antibody specific for the Kappa light chain of mouse, rat, or rat/hamster Ig, rather than cells, to perform the single staining controls according to the manufacturer’s instructions. See Note 9. 3. Fluorescence Minus One (FMO) controls. Stain cells with all the fluorochromes of your panel but one. Prepare an FMO for each fluorochrome. These controls are helpful to identify and gate cells in the context of data spread due to the multiple fluorochromes. See Table 1 and Note 10.

3.4

Staining of Cells

In order to compare different samples, it is important that all the samples: – have been processed in the same way – contain a very similar number of cells – are stained at the same time, with the same concentration of antibodies, preferably the same antibodies mix.

Table 1 Example of FMO controls needed for a 4-color panel FMO control

FITC

PE

PE/Cy7

APC

FITC FMO

–

PE Ab

PE/Cy7 Ab

APC Ab

PE FMO

FITC Ab

–

PE/Cy7 Ab

APC Ab

PE/CY7 FMO

FITC Ab

PE Ab

–

APC Ab

APC FMO

FITC Ab

PE Ab

PE/Cy7 Ab

–

Flow Cytometry for Beginners 3.4.1 For Surface Protein Staining (Where Permeabilization Is Not Required)

35

1. Adjust cell concentration to 1–10 millions/mL (see Note 4). 2. Aliquot 100μL of cells in PBS 0.5% Bovine Serum Albumin (BSA) in each tube and remember the negative controls and, if you have one, a positive control (see Subheading 3.3.). If, for instance, you are staining your cells with N ¼ 5 antibodies, remember to prepare (5
2) + 1 extra tubes for controls (1 unstained sample, 5 single-stained controls, and 5 FMO samples). 3. Centrifuge briefly to collect the cells at the bottom. 4. Carefully remove the PBS. 5. Optional (see Note 11), dilute Fc block 1 to 50 in PBS and add 100μL to each sample. Incubate for 20 min on ice. Centrifuge at 400
g for 5 min at 4  C. Gently discard the supernatant and resuspend the cells in 100μL of PBS 0.5% BSA. 6. Prepare a mix of PBS for all the samples with all the antibodies (0.1–10μg/mL of each primary labeled antibody). Resuspend the cells in the mix and incubate for at least half an hour at room temperature or at least 1 h on ice in the dark (covered by aluminium foil). 7. Wash the cells twice with 500μL of PBS 0.5% BSA. 8. Resuspend the cells in fresh PBS for acquisition. 9. Keep the samples on ice in the dark until acquisition. 10. For viability dye, see Note 1. 11. Acquire samples as soon as possible. 12. If you need to keep the cells for days, after step 7, fix your cells by resuspending the samples in 4% paraformaldehyde and incubate for 10–15 min at room temperature. 13. Wash the cells with 500μLof PBS. 14. Resuspend the cells in ~200μL cold PBS and store them at 4  C until needed.

3.4.2 For Intracellular Staining

Intracellular staining can be performed to detect intracellular proteins, to analyze signal transduction upon a stimulus, or to detect cytokine production. See Note 12. This analysis requires cell fixation and permeabilization before staining, typically using formaldehyde to preserve the cellular morphology, followed by permeabilized with detergent or alcohol. Many companies produce kits for cell fixation and permeabilization. Here, we give a general protocol not using these commercial kits. 1. Prepare cell suspensions as described in Subheading 3.1. 2. Perform staining of cell surface antigens as described in Subheading 3.4.1. (Optional) and then resuspend cells in 100μL PBS with 0.5% BSA. See Note 13.

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3. To exclude dead cells from analysis, resuspend cells in PBS 0.5% BSA and stain with a fixable dead cell dye, according to manufacturer’s instructions (Optional). Wash cells with PBS 0.05% BSA and resuspend them in 100μL PBS with 0.5% BSA. 4. Add 100μL of a 4% paraformaldehyde solution prepared in PBS and incubate for 15 min at room temperature. See Note 14. 5. Wash fixed cells twice with PBS 0.5% BSA. Centrifuge at 400
g for 5 min at 4  C. Gently discard the supernatant and resuspend the cells in 100 μL of PBS 0.5% BSA. 6. Permeabilize cells by adding 1 mL of 0.1% saponin in PBS 0.5% BSA and incubating for 15 min. See Note 15. 7. Centrifuge at 400
g for 5 min at 4  C. Gently discard the supernatant and wash cells with PBS 0.5% BSA plus 0.1% saponin (in order to maintain permeabilization during the wash steps). Centrifuge at 400
g for 5 min at 4  C. Gently discard the supernatant and resuspend the cells in 100 μL of PBS 0.5% BSA. 8. Incubate with primary antibody for 30 min at room temperature. 9. Wash twice with PBS 0.5% BSA 0.1% saponin. 10. If the primary antibodies were not directly labeled with fluorochromes, add fluorochrome-conjugated secondary antibody and incubate for 30 min at room temperature. 11. Wash twice with PBS 0.5% BSA 0.1% saponin. 12. Resuspend the cells in ~200 μL cold PBS and store them at 4  C until needed or acquire them. 13. Before acquiring the cells, stain them with live/dead staining dyes. See Note 1 and Table 2. 3.5 Instrument Setup and Data Acquisition

1. Before starting the analysis, perform daily instrument setup and calibration by using calibration beads, such as CS&T beads (BD Biosciences) or 8-peak Rainbow bead calibration particles (Spherotech), according to instrument’s and manufacturer’s instructions. This procedure allows to set a baseline voltage for each detector, to standardize experimental setup, and to monitor the instrument performance over time [3]. 2. Optimize PMT voltage. Acquire unstained and stained cells and check if the positive population falls in the linear range of the detector, see Note 16. 3. Acquire the compensation controls and perform compensation to correct fluorescence spillover. Fluorescence spillover is the amount of signal (measured as median fluorescence intensity) that a fluorochrome emits in a detector specific for another fluorochrome. This spillover can be subtracted using a

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compensation matrix based on the acquisition of single color controls. Many flow cytometer softwares have automatic compensation algorithms that usually give optimal results. However, the user should verify that the software is providing correct gates and results. 4. Acquire the negative controls and the FMO controls and design the gating strategy for your experiment in order to correctly identify the different cell populations present in your samples. 5. Apply a threshold to remove small debris and distinguish the signal (using fluorescence or scatter) required to define the population of interest from the background. 6. Use a time plot to eliminate the spurious events due to clogs or transient problems during acquisition. Include doublet discrimination plots and exclude dead cells (on the base of vitality dyes). 7. Design a series of sequential gates on the different fluorescent markers included in the experiment to discriminate between positive and negative cells and specifically identify the different cell types. The best way to determine the fluorescence gating strategy is to use FMO controls to set the population negative for the different markers. See Note 10. 8. Once the instrument has been setup and all the controls acquired, proceed with the acquisition of the experimental samples. The number of events that should be acquired to obtain significant data is not a fixed value, but depends on several experimental features such as the frequency of the cell population of interest and the number of markers used in the experiment. It can be calculated by applying Poisson statistics. However, in general, for rare populations, as many events as possible should be acquired. See Note 17. 3.6

Data Analysis

The .FCS files generated by the flow cytometer can be analyzed either using the instrument’s software or using third parties’ softwares such as FlowJo. Data analysis can be performed either manually or using computational techniques. Although automatic analysis is particularly useful to handle large and/or highdimensional data sets, manual analysis is still extensively used to identify cell subpopulations and quantify the expression of specific markers. Manual analysis allows the visualization of data in univariate histograms or bivariate plots that can be represented by either dot plots, density plots, or contour plots. Here, a simple procedure for manual analysis based on sequential gating on bivariate plots is reported. Rectangular gates can be used for well-separated subpopulations, but more subtle gates are often required to define near subpopulations or due to compensation fluorescence spreading.

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1. Perform flow stability gating by plotting one parameter (for example, FSC) in function of time and draw a gate excluding events acquired when the flow was not stable (for example, in the first seconds of acquisition or if a clog was acquired). 2. Display scatter properties of your sample in a bivariate plot and gate the desired cell population based on its forward and side scatter characteristics, thus excluding debris and other cell populations. 3. Perform doublet exclusion using pulse geometry gating by plotting the height or width against the area for either forward or side scatter. 4. Exclude dead cells by plotting the viability dye against a scatter channel. 5. Display the selected events of density plots displaying cell lineage markers and perform sequential gating on the different cell populations. FMO samples should be used to discriminate between positive and negative cells (see Fig. 2, showing CD4+ T cell activation in a mouse spleen [4]). 6. Optimize the gates using backgating to confirm a staining pattern or gating method. This technique allows for the visualization of the cells in the final gate at higher levels of the gating strategy, to check if the gates are correctly positioned. 7. When possible, examine the final gated subpopulations carefully, using prior knowledge and positive and negative controls, to check the correctness of the analysis. 8. In most analysis softwares, you can export to Excel or to other similar softwares many data such as the percentage of each cell subpopulation, the levels of expression of the different markers in the different subpopulations or their distribution pattern. These numerical data can be used to perform statistical analysis.

4

Notes 1. In order to perform a correct data analysis, it is important to discriminate between live and dead cells within each sample. This is mainly due to the fact that dead cells are often highly autofluorescent and they show higher nonspecific antibody binding, leading to a significant percentage of false-positive events. To overcome this limitation, on top of the forward and side scattering gating strategy, many dyes are available to distinguish live from dead cells. This so-called dead/live staining dyes belong to two main categories: the nucleic acid binding dyes that selectively stain dead cells as they are not membrane permeable so can enter only in dead cells whose membrane are compromised, and protein binding dyes that

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39

stain both live and dead cells with dead cells being much brighter due to the same mechanism. The most used DNA staining are DRAQ7, DRAQ5, Propidium iodide, 7-AAD, TO-PRO-3, and Hoechst 33258 (see Table 2). All of these are added to the samples just before acquisition, and they can’t be used if fixation is needed (or on fixed samples, of course). For experiments where it is crucial to be able to stain for live/dead cells and preserve this pattern after fixation, few options are available including the Invitrogen Molecular Probes LIVE/DEAD Fixable Dead Cell Stains and the Biolegend Zombie Dyes, all of them available in several flavors. Dyes can be added to samples just before fixation. 2. Trypsin cleaves the focal adhesions that are anchoring the cell to the culture dish. It can be used at different concentrations ranging from 0.25 to 0.05%. Timing and temperature also affects its efficiency. For most cancer cell lines between 1 and 3 min at 37  C will be enough to completely detach cells from the plate and also to disrupt cell to cell contact. Pay attention that too high trypsin concentration or a too long incubation can kill the cells. Also pay attention that it does not damage the antigen you are going to analyze. 3. Adding 25 mM HEPES or 0.05/0.1 mM EDTA in the buffer will prevent cells from forming aggregates again. Keep the cells on ice throughout the procedure will also help. 4. The number of cells must be roughly between 100.000 and 1 million per sample. 5. Collagenase time and temperature must be determined for different tissues. For tissues rich in extracellular matrix but with relatively fewer cells 15 min on ice is enough. Other tissues will need 30 min at 37 . Some tests will be needed to optimize the right time and temperature. 6. Ideally, fluorophores with no or little emission spectra overlap should be chosen. However, in complex multicolor panels this is challenging. In this case, fluorochromes with the narrower excitation and emission spectra should be preferred. 7. Many antibody suppliers have compiled published information about antigen expression in various cell types. These antigen density charts can be found on their websites. Moreover, panel building programs are now available online to help with panel building. The most used are Chromocyte (@https://www. chromocyte.com/) and FluoroFinder (@https://fluorofinder. com/). 8. To perform titration, stain a fixed number of cells in a fixed volume of buffer, for a fixed incubation time and temperature, with scalar doses of antibody. Calculate the Stain Index (SI) for each sample (the difference between the mean fluorescence

40

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intensity of the positive and negative populations, divided by two times the standard deviation of the negative populations). Then, plot the obtained SI on the antibody concentration and identify the concentration that gives the best SI [5]. 9. The compensation samples should be as bright as possible; the background of the negative and positive populations should be similar; the fluorochrome must be the same in compensation and experimental samples. When using tandem dyes, consider that dyes from different vendors or different batches can have different features and amount of spillover. So, a separate singlestained control should be used for each of these dyes. In some cases, beads do not work as perfect surrogates of cells. When working with particular biological samples or testing an antibody for the first time, it could be useful to compare singlestained samples performed on beads and with the cells of interest, to verify whether they give similar results. If this does not happen, use cells as compensation control. If possible, acquire at least 5000 positive and 5000 negative events to assure accurate measurements [6]. 10. In the past, gates were set on the basis of the signal of isotype control-stained samples. However, these antibodies may have different non-specific binding properties than the antibodies we are using, thus not allowing to really discriminate between positive and negative events. These controls can only be used to identify potential blocking problems. 11. Despite this step is optional, we recommend to perform it if your cells express a high level of Fc receptor that will increase the non-specific binding and the background fluorescence. 12. For the analysis of cell signaling or cytokine production, activated cell populations can be prepared from in vivo-stimulated tissues or from in vitro-stimulated cultures (using receptor ligands or, for T cell activation, antigen-specific, or mitogeninduced activation). For cytokine and chemokine analysis, it is critical to incubate cells with a protein transport inhibitor such as brefeldin A or monensin in the last 4–6 h of cell culture [7]. 13. It is better to perform surface staining before cell fixation and permeabilization to avoid any potential effects of the intracellular staining protocol on surface antibody binding. 14. If 4% paraformaldehyde does not work, try fixing with lower concentrations as this may help to preserve your epitope. 15. Saponin may be substituted by other permeabilization agents such as Digitonin, Triton X-100, or NP-40. For nuclear or phosphorylated antigens, fixation may be followed by incubation with 90% ice-cold methanol for 30 min at 4  C, followed by two washes and antibody staining. In the case of methanol, methanol-resistant fluorochromes such as FITC and AlexaFluor dyes should be used.

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16. The PMT voltage defined during instrument calibration can be usually used to acquire the experimental samples. However, in some cases further optimization may be required. If highly stained cells are above the linear maximum or off scale, the PMT voltage should be reduced. If the cell population has a different autofluorescence than the calibration beads, run unstained cells, and adjust the PMT gain [6]. In BD instruments, the optimized PMT voltages can be saved in reference to CS&T baseline values. In this way, they are automatically modified in reference to any ΔPMT between CS&T baseline and CS&T daily performance checks. 17. For very rare populations that need the acquisition of high numbers of events, the best practice is to collect each sample in separate files containing 1–2 million events, and then concatenate these files into a unique one using analysis softwares. Moreover, parameters that are not needed in the experiment can be unselected to minimize the file size.

Acknowledgements Valentina Proserpio is supported by Fondazione Umberto Veronesi -FUV-. This work was supported by the University of Torino. References 1. Herzenberg LA, Parks D, Sahaf B, Perez O, Roederer M, Herzenberg LA (2002) The history and future of the fluorescence activated cell sorter and flow cytometry: a view from Stanford. Clin Chem 48:1819–1827 2. Conti L, Bolli E, Di Lorenzo A, Franceschi V, Macchi F, Riccardo F, Ruiu R, Russo L, Quaglino E, Donofrio G et al (2020) Immunotargeting of the xCT cystine/glutamate antiporter potentiates the efficacy of HER2-targeted immunotherapies in breast cancer. Cancer Immunol Res 8:1039–1053 3. van der Strate B, van der Strate B, Longdin R, Geerlings M, Bachmayer N, Cavallin M, Litwin V, Patel M, Passe-Coutrin W, Schoelch C et al (2017) Best practices in performing flow cytometry in a regulated environment: feedback from experience within the European bioanalysis forum. Bioanalysis 9:1253–1264 4. Donofrio G, Tebaldi G, Lanzardo S, Ruiu R, Bolli E, Ballatore A, Rolih V, Macchi F,

Conti L, Cavallo F (2018) Bovine herpesvirus 4-based vector delivering the full length xCT DNA efficiently protects mice from mammary cancer metastases by targeting cancer stem cells. Onco Targets Ther 7:e1494108 5. Maecker HT, Frey T, Nomura LE, Trotter J (2004) Selecting fluorochrome conjugates for maximum sensitivity. Cytometry A 62:169–173 6. Cossarizza A, Chang H-D, Radbruch A, Acs A, Adam D, Adam-Klages S, Agace WW, Aghaeepour N, Akdis M, Allez M et al (2019) Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition). Eur J Immunol 49:1457–1973 7. Conti L, De Palma R, Rolla S, Boselli D, Rodolico G, Kaur S, Silvennoinen O, Niccolai E, Amedei A, Ivaldi F et al (2012) Th17 cells in multiple sclerosis express higher levels of JAK2, which increases their surface expression of IFN-γR2. J Immunol 188:1011–1018

Chapter 4 High-Dimensional Immunophenotyping with 37-Color Panel Using Full-Spectrum Cytometry Marco A. Fernandez, Hammad Alzayat, Maria C. Jaimes, Yacine Kharraz, Gerard Requena, and Pedro Mendez Abstract A comprehensive study of the cellular components of the immune system demands both deep and broad immunophenotyping of numerous cell subsets in an effective and practical way. Novel full-spectrum technology reveals the complete emission spectrum of each dye maximizing the amount of information that can be obtained on a single sample regarding conventional flow cytometry and provide an expanded knowledge of biological processes. In this chapter, we describe a 37-color protocol that allows to identify more than 45 different cell populations on whole blood samples of SARS-CoV-2-infected patients. Key words Full-spectrum cytometry, Deep immunophenotyping, Spectral signature

1

Introduction Besides microscopy, the first technology capable of measuring and analyzing cell properties at the single-cell level is flow cytometry (FC). In 1976, Leonard A. Herzenberg, published the blueprint of the first FACS instrument, using one laser and two light detectors [1]. Ever since, the field of immunology has continuously been pushing the development of flow cytometry devices. The discovery and integration of new and brighter fluorophores, their conjugation on antibodies, incorporation of multiple lasers, improved optic systems, and advanced software development, nowadays allows the detection and accurate quantitation of 25+ parameters by FC [2, 3]. However, a further increase in plexy represents a challenge due to the overlap of excitation and emission peaks of fluorophores. The current outbreak caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus is the most critical

The original version of this chapter was revised. The correction to this chapter is available at https://doi.org/ 10.1007/978-1-0716-1771-7_18 Aik T. Ooi (ed.), Single-Cell Protein Analysis: Methods and Protocols, Methods in Molecular Biology, vol. 2386, https://doi.org/10.1007/978-1-0716-1771-7_4, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022, Corrected Publication 2022

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public health [4] and socio-economic [5] problem worldwide in 2020. Despite its relatively low global mortality rate (~2.5%) [6], it is a highly transmissible virus, with heterogeneous patient outcomes and potential sequels post-infection. To identify biomarkers of disease prognosis and prediction of sequels, an extensive immunophenotypic study of infected patients with mild or no symptoms, and patients with poor outcomes, is necessary. The full-spectrum cytometry represents the natural technological evolution of FC [7]. A fluorophore is identified by its complete emission spectra over each laser, instead of just its excitation and emission peaks. It allows an increase in the number of fluorophores and, thus, parameters assayed. Beyond detection and quantitation purposes, further technology developments will allow to physically sort cell subpopulations from high-dimensional data generated by full-spectrum cytometry. The new platform is expected to be released anytime in spring or summer of 2021. In the current protocol, we measure 37 parameters using the Cytek Aurora 5-laser spectral system for a multi-dimensional immunophenotype of SARS-CoV-2-infected patients.

2

Materials

2.1 Buffers and Reagents

1. Red blood cells (RBC) lysis buffer stock solution (10X): Mix 80.2 g NH4Cl (1.5 M), 8.4 g NaHCO3 (100 mM), 3.7 g disodium EDTA (10 mM), in 900 mL of ddH2O. Adjust pH to 7.4 with 1 N HCl or 1 N NaOH. Adjust the volume with ddH2O up to 1 L. Store
6 months at 4  C. For 1X solution, dilute 10X solution with deionized water (store at room temperature). Alternatively, commercial solutions of 10X ammonium lysis buffer could be used. 2. Phosphate-buffered saline (PBS). Store at room temperature. 3. Paraformaldehyde (PFA) working solution: 1% PFA in 1X PBS. Store at 4  C. 4. Stain/wash buffer: 2% fetal bovine serum and 0.02% sodium azide solution in 1X PBS. Store at 4  C. 5. Brilliant Stain Buffer Plus (BD Biosciences). Store at 4  C. 6. True-Stain Monocyte Blocker (BioLegend). Store at 4  C. 7. Sheath fluid (FACSFlow, BD Biosciences). Store at 4  C. 8. Viability dye (a) Stock solution: add 50 μL of DMSO to a vial of LIVE/DEAD™ Fixable Blue Dead Cell Stain Kit for UV excitation (Thermo Fisher). Mix well and visually confirm that all of the dye has dissolved. Make 5 μL aliquots and store at 20  C. (b) Working solution: thaw and dilute stock solution 1:40 in 1X PBS. Store at 4  C, protected from light until use (within 1 h). Discard unused fraction. 9. SpectroFlo® QC Beads (Cytek Bioscences). Store at 4  C.

37-Color Immunophenotyping Using Full-Spectrum Cytometry

2.2 Monoclonal Antibodies

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1. Antibodies panel (Table 1) 2. Chemokines Master Mix (Table 2) 3. General Antibodies Master Mix (Table 3)

2.3 Consumables and Equipment

1. 50 mL and 15 mL conical polypropylene tubes 2. 5 mL round-bottom polypropylene tubes 3. Rocking platform 4. Vortex 5. Centrifuge for 50, 15, and 5 mL tubes with sealing caps. 6. Biosafety Cabinet Class II 7. Cytek Aurora 5-laser Spectral Cytometer

3

Methods This method was developed on a 5-laser Cytek Aurora spectral cytometer. Some combinations of selected fluorochromes in this panel (Table 1), like FITC/BB515 or APC/Alexa647 only can be resolved if a full spectral cytometer is used as the whole spectrum is analyzed. The method outlined in this chapter is based on a protocol and 35-color panel originally designed by Cytek Biosciences R&D Department in PBMCs and recently was published for a 40-color panel (see Notes 9 and 10). Modifications were made to suit the specific needs of the panel and to optimize the performance of whole blood samples. For fluorochrome selection and panel design, two indexes developed by Cytek Biosciences were used. All selected fluorochromes must have unique spectra when compared across the 64 detectors. The similarity index uses the overlay of dyes spectra to determine their degree of similarity. This index ranges from 0 to 1; 0 indicates that two spectra are completely different while 1 means spectra match perfectly. The Complexity Index is an overall measurement of uniqueness of the dyes used in the panel (Fig. 1). The lower the complexity index is, the easier the combination of dyes to work with. Fluorochrome-antibody pairs were selected following previously published approaches [8]. Briefly, antigens were classified by level of expression on subsets of interest (primary, secondary, and tertiary). Low-expression antigens are assigned to a high index stain fluorochrome. Moreover, availability of conjugated antibodies must be considered. The steps of method development and validation includes: (a) Individual titration of each antibody or viability dye, prior testing samples of interest [9]. It includes the standardization of volumes of cell staining and pellet resuspension prior to spectral cytometry analysis.

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Table 1 Monoclonal antibodies used in 37-color panel Specificity

Fluorochome

Vendor

Clone

Main purpose

CD45RA

BUV395

BD Biosciences

5H9

T cell subsets

CD16

BUV496

BD Biosciences

3G8

NK, monocytes, and DC

CD195 (CCR5)

BUV563

BD Biosciences

2D7

Chemokine receptor

CD24

BUV615

BD Biosciences

ML5

B cells subsets

CD11c

BUV661

BD Biosciences

B-ly6

DC differentiation

CD62L

BUV737

BD Biosciences

SK11

Neutrophil activation

CD8

BUV805

BD Biosciences

RPA-T8

CD8 T cell, NK cell

CD197 (CCR7)

BV421

BioLegend

G043H7

T cells subsets

CD123

Super Bright 436

Thermo Fisher

6H6

Plasmacytoid dendritic cells

CD161

eFluor450

Thermo Fisher

HP-3G10

T and NK cell subsets

IgD

BV480

BD Biosciences

IA6-2

B cell differentiation

CD3

BV510

BioLegend

SK7

T cells

IgM

BV570

BioLegend

MHM-88

B cell differentiation

IgG

BV605

BD Biosciences

G18-145

B cell differentiation

CD28

BV650

BioLegend

CD28.2

T cell subsets

CD196 (CCR6)

BV711

BioLegend

G034E3

Chemokine receptor

CXCR5 BV750 (CD185)

BD Biosciences

RF8B2

Chemokine receptor

CD279 (PD-1)

BV785

BioLegend

EH12.2H7 T cell inhibitory receptor

IgA

FITC

Miltenyi

REA1014

B cell differentiation

CD11b

BB515

BD Biosciences

ICRF44

Monocyte and DC subsets

CD14

SparkBlue550

BioLegend

63D3

Monocyte

CD45

PerCP

BioLegend

2D1

Leukocyte

TCR Vδ2

PerCPVio700

Miltenyi

REA771

TCRgd T cell subset

CD194 (CCR4)

BB700

BD Biosciences

1G1

Chemokine receptor

TIGIT

PE

BioLegend

A15153G

T cell inhibitory receptor

CD4

cFluor YG584 Cytek

SK3

CD4 T cells (continued)

37-Color Immunophenotyping Using Full-Spectrum Cytometry

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Table 1 (continued) Specificity

Fluorochome

CD57

Vendor

Clone

Main purpose

PE-Dazzle594 BioLegend

SN3

Senescence T and NK cell

CD95

PE-Cy5

BioLegend

DX2

T and B cell subsets

CD25

PE-AF700

Thermo Fisher

3G10

Activated and regulatory T cells

CD183 PECy7 (CXCR3)

BioLegend

G025H6

Chemokine receptor

CD27

APC

BioLegend

M-T271

T and B cells subsets

CD56

AlexaFluor647 BD Biosciences

R19-760

NK cell

CD19

SparkNIR685

BioLegend

HIB19

B cell

CD127

APC-R700

BD Biosciences

HIL-7RM21

T cell subsets

HLA-DR

APC-Fire750

BioLegend

L243

T, B, NK, DC, and monocyte subsets

CD38

APC-Fire810

BioLegend

HIT2

Activated T and B cells, monocyte, and DC differentiation

Table 2 Master Mix of chemokines receptor staining Chemokines Master Mix (add in this order) Item

Volume (μL/test)

Brilliant Stain Buffer Plus Stain/wash Buffer

10.0

a

31.3

PECy7 anti-human CD183 (CXCR3)

2.5

BUV563 Mouse anti-human CD195 (CCR5)

2.5

BB700 Mouse anti-human CD194 (CCR4)

2.5

Brilliant Violet 711™ anti-human CD196 (CCR6)

1.2

Stain/wash buffer is used to ensure 50 μL of final volume. Titer volume shown is for one test sample

a

(b) Compare staining performance of combined antibody cocktail with the individual titration experiments. (c) Test the minimum number of cells required to detect rare cell populations accurately. Use a sample known to contain the targeted rare cell population/s of interest, with a decreasing number of cells per reaction (e.g., 5  106, 2.5  106, 1.25  106, 0.625  106) The cells were resuspended in a final volume of 250 μL to mimic the final staining volume in all antibodies tube.

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Table 3 General antibodies Master Mix Item

Volume (μL/test)

CD161 Monoclonal Antibody (HP-3G10), eFluor 450

5

PE anti-human TIGIT (VSTM3) Antibody

5

APC anti-human CD27 Antibody

5

®

Alexa Fluor 647 Mouse anti-human CD56

5

PE-Alexa Fluor 700 anti-human CD25 Antibody

2.5

CD123 Monoclonal Antibody (6H6), Super Bright 436

2.5

Spark Blue™ 550 anti-human CD14 Antibody

2.5

APC-R700 Mouse anti-human CD127

2.5

APC/Fire™ 750 anti-human HLADR

2.5

APC-Fire810 CD38

1

PerCP anti-human CD45 Antibody

1

PE/Cy5 anti-human CD95 (Fas) Antibody

0.6

CF568 CD4

0.5

PE/Dazzle™ 594 anti-human CD57 Antibody

0.1

Brilliant Stain Buffer Plus

10

Intermediate mixing by pippeting BUV737 Mouse Anti-Human CD62L

0.3

BUV496 Mouse Anti-Human CD16

0.6

CD8 BUV805

0.6

BV480 Mouse Anti-Human IgD

1

BUV395 Mouse Anti-Human CD45RA

1.2

BD Horizon™ BB515 Mouse Anti-Human CD11b

1.2

BUV661 Mouse Anti-Human CD11c

2.5

Brilliant Violet 510™ anti-human CD3

2.5

Brilliant Violet 570™ anti-human IgM Antibody

2.5

Brilliant Violet 650™ anti-human CD28 Antibody

2.5

BD Horizon™ BV605 Mouse Anti-Human IgG

2.5

BUV615 Mouse Anti-Human CD24

5

Brilliant Violet 785™ anti-human CD279 (PD-1) Antibody

5

Vortex and spin Volume (μL/test) a

Antibodies excluded in alternative protocol. Titer volume shown is for one test sample

73.1

37-Color Immunophenotyping Using Full-Spectrum Cytometry

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Fig. 1 All fluorochromes of 37-color panel displayed on Cytek Full Spectrum Viewer (https://spectrum.cytekbio. com/). Overall panel Complexity Index: 42.33

(d) Reproducibility of the assay. Once the optimal number of cells required in the assay is known, perform three independent experiments, in triplicates, each experiment. It will help to understand the intra- and inter-experimental variability. We set a final staining volume of 235 μL and a total of 5  106 cells, per reaction. The current antibody panel has been designed to study the immunological profile of peripheral blood mononuclear cells (PBMCs) from SARS-CoV-2 patients. However, it can also be applied to analyze the immunophenotype of immune diseases or circulating tumor cells at single-cell and population level [10, 11]. CDC recommends to manipulate SARS-CoV-2 specimens under a Biosafety Level 3 laboratory, using BLS-3 practices [12]. 3.1 Lysis of Red Blood Cells (see Note 1)

1. Pipette 27 mL of freshly made 1X RBC lysis buffer into a 50 mL conical tube. 2. Transfer 3 mL of well-mixed blood to the tube prepared in step 1. Final ratio 1:9 of blood:1X RBC lysis buffer. 3. Cap the tube, mix gently by inversion, and place on a rocking platform for 5 min at medium speed. 4. Centrifuge at 400 g for 5 min at room temperature. 5. Discard the supernatant without disturbing the pellet. 6. Add 1 mL of 1X RBC lysis buffer to the tube, mix by pipetting. Transfer to a 15 mL tube conical tube with 9 mL of 1X RBC lysis buffer, mix by pipetting, and place on a rocking platform for 2 min, at medium speed. 7. Repeat steps 4 and 5. 8. Add 1 mL of stain/wash buffer, mix by pipetting. Then add 9 mL of stain/wash buffer.

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9. Repeat steps 4 and 5. 10. Add 1 mL of stain/wash buffer and mix by pipetting. 11. Count cells. For direct counting on Aurora spectral cytometer, see Note 2. 3.2

Cell Staining

3.2.1 Dead Cells Staining

1. For a given sample, pipette 5  106 cells into a clean 5 mL FACS tube (polypropylene recommended). 2. Add 3 mL of PBS. For comments about number of cells, see Note 3. 3. Centrifuge 400 g for 5 min at room temperature. 4. Decant supernatant without disturbing pellet (pellet final volume  60 μL approx.). 5. Add 40 μL of 1x PBS for an approximate volume of 100 μL. Mix by gentle vortexing. 6. Add 5 μL of viability dye working solution and vortex. 7. Incubate for 15 min. at RT in the dark. 8. Add 3 mL of stain/wash buffer. 9. Centrifuge 400 g for 5 min at room temperature. 10. Decant supernatant without disturbing pellet (pellet final volume  60 μL approx.) 11. Add 40 μL of stain/wash buffer for an approximate volume of 100 μL. Mix gently by vortexing. 12. Keep in dark (room temperature) until ready to use (within 1 h).

3.2.2 Immunostaining

1. Add 5 μL True-Stain Monocyte Blocker™ and 5 μL of Brilliant Stain Buffer Plus, vortex for 5 s (Note 4). 2. Add 5 μL of anti-CCR7 and 1.2 μL of anti-CXCR5 antibodies (Note 5). 3. Vortex then incubate at RT for 10 min protected from light. 4. Add 50 μL of the Chemokines Master Mix (Table 2). Mix by pipetting. 5. Vortex and incubate at RT for 15 min protected from light. 6. Add 73.1 μL of the General Antibodies Master Mix (see Note 6; Table 3). Mix by pipetting. 7. Incubate at RT protected from light for 20 min. 8. Add 3 mL of stain/wash buffer to each tube. Vortex for 5 s. 9. Centrifuge at 400 g for 5 min at room temperature. 10. Decant supernatant. 11. Repeat steps 9 and 10. 12. Add 300 μL of PFA 1% in PBS. Mix by pipetting.

37-Color Immunophenotyping Using Full-Spectrum Cytometry

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13. Incubate for 15 min at room temperature protected from light. 14. Repeat steps 9 and 10. 15. Add 40 μL of stain/wash buffer, 1 μL of CD19 SparkNIR685, 1 μL of TCR V2d, and 1 μL of IgA antibodies (see Note 7). 16. Vortex and incubate for 15 min. 17. Resuspend the cell pellet with 600 μL of Sheath Fluid/FACSFlow reagent. Vortex for 5 s. 18. Run the sample tube in spectral cytometer or store at 4  C protected from light (see Note 8). 3.3 Spectral Cytometer Setup and Data Analysis

1. Apply Cytek Assay Settings for acquisition configuration and run SpectroFlo® QC Beads as recommended by the manufacturer (see Note 9). 2. Single and unstained controls are required for spectral unmixing (see Note 10). 3. Analyze data using conventional manual gating or preferably by dimensional reduction and clustering algorithms (see Note 11).

4

Notes 1. Our panel was designed to monitor the immune system in whole blood from SARS-CoV-2 patients, and it is optimized for low concentration of MNCs [13]. If the sample’s source is either buffy coat, leukopak, or PBMCs obtained by density gradient (Ficoll), the RBC lysis step, can be omitted. Three mL of fresh whole blood obtained in EDTA tubes provides enough MNCs for the analysis. Lower volume of whole blood could be applied if it doesn’t compromise the detection of rare cell populations. Adjust the volume of 1x RBC lysis buffer according to the starting volume of whole blood. 2. Aurora spectral cytometer allows direct volumetric count. For counting cells using Aurora spectral cytometer, mix 25 μL of sample and 175 μL of Sheath Fluid/FACSFlow buffer (both by reverse pipetting). Acquire data on Aurora. To calculate the cell concentration, multiply the number of “cells/μL” (statistic of gated “leukocytes”), by its dilution factor (dilution factor ¼ 8). 3. As lymphopenia has been associated with severe COVID-19 samples [13], 5  106 cells are required for the all antibodies staining tube to ensure a minimal number of events detection in low frequency subsets. 4. True-Stain Monocyte Blocker™ is used to avoid nonspecific binding of cyanine-based fluorochromes like PE-Cy5, PE-Cy7, PerCPcy5.5, APC-Fire750, or PE-Dazzle594 to monocytes

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and macrophages. Adding Brilliant Stain Buffer Plus buffer to the tubes and cocktails is mandatory to reduce staining artifacts when using multiple BD Brilliant fluorochrome reagents. 5. Based on previous reports [14], sequential staining of chemokine receptor improves their detection when multiple receptors are stained in the same panel. Several combinations were evaluated and empirically noticed that an initial staining of antiCCR7 and CXCR5 antibodies, prior to the immunostaining of the rest of the chemokines improved their detection, specifically for CXCR5 on T cells. A master mix of all the remaining chemokine receptors antibodies (Table 2) are added after CCR7 and CXCR5 staining step and before adding the general antibodies cocktail. 6. In the general antibodies mix cocktail, Brilliant Stain Buffer Plus is needed to reduce interactions between Brilliant fluorochromes as indicated in Note 4. All but chemokine receptors, CD19, TCRVd2, and IgA are included in this mixture. Add first all non-Brilliant fluorochromes to get a minimal volume of 50 μL and then add the Brilliant dye-conjugated antibodies. As more than one Brilliant Ultraviolet (BUV) reagent are included in the cocktail, mix must be used within 2 h after preparation. As a general rule, it’s also recommended that every new lot of antibody be tittered. 7. Optimization of panel staining is an essential step for any multicolor cytometry assay. Designing a large panel requires a thorough validation and optimization to identify and correct potential cross-reactivity, unspecific staining, and artifacts of antibodies before analyzing clinical samples from patients [8, 15]. During the development of the panel presented in this protocol, it was necessary some adjustments to overcome some artifacts. As an example, nonspecific staining was detected between anti-IgG, anti-TCR Vd2 and anti-IgA antibodies due to the binding of IgG antibody to both TCRVd2 and IgA antibodies (see Fig. 2a). To avoid this issue, the original protocol was modified placing the staining of TCRVd2 and IgA in the step after PFA’s fixation (see Fig. 2b). Another adjustment was required for the anti-CD19 antibody. Cleaner signal-to-noise was achieved in detecting CD19+ cells, when anti-CD19 staining was performed after the fixation step (see Fig. 3). 8. Incubation of CD19, TCRVd2, and IgA without washing step improves signal to noise ratio if acquisition is performed within 1 h after staining. If acquisition is delayed more than 1 h, an additional washing step should be added. Because several cellular subsets are present at low frequency, and in addition COVID patients have a marked lymphopenia, a minimum of

37-Color Immunophenotyping Using Full-Spectrum Cytometry

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Fig. 2 Anti-IgG antibody cross-reacts with anti-TCR Vd2 (A) and anti-IgA (B). Left plots, standard protocol with simultaneous staining; right plots, alternative staining with sequential staining

3  106 events corresponding to the whole peripheral blood cellularity must be acquired to obtain information on at least of 50.000 PBMCs cells. 9. On the Aurora spectral cytometer, the “Cytek Assay Settings” are optimized to provide improved gains for all detectors. This optimization has been done by Cytek R&D Department [16]

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Fig. 3 CD19 SparNIR685 signal pre- and post-fixation step. Post-fixation staining shows better resolution of CD19+ cells

to provide the best resolution of most known fluorophores on antibody-stained cells focused on final multicolor tube. It was generated by measuring the optimal resolution of human lymphocytes stained with CD8 and CD4 labeled with several fluorochromes. These settings provide a starting point for most immunophenotyping applications. Gains are automatically adjusted after each daily QC based on laser and detector performance towards a target value, which is defined by the detection and optimal assignment of proprietary fluorescent beads against predetermined target values. It’s highly advisable to run a newly/fresh tube with QC control beads (SpectroFlo® QC Beads, Cytek) before acquiring samples to guarantee that accurate gain settings will be applied to experiment. 10. As for spectral compensation, accuracy of the data is dependent on controls and their ability to represent the spectra of fluorochromes used in the panel. Reference controls, obtained by running single-stained and unstained samples, provide the individual fluorescence spectra necessary to unmix the spectral signature of each fluorochromes used on the panel [16]. Single and unstained controls should be treated the same way as the fully stained samples. The use of cells as the experimental sample is desirable than control beads although they match the fluorochrome spectral properties. During the unmixing procedure, a mathematical algorithm (Ordinary Least Squares, OLS) is used for the decomposition of the fluorescent components in the sample using the reference controls. Cellular autofluorescence can also be treated as another fluorochrome in this process allowing extraction that markedly improved the overall signal resolution. Data was unmixed in real-time or postacquisition with the SpectroFlo software v2.2 (Fig. 4). 11. Although is not the purpose of this chapter, analysis of data obtained from a high-dimensional panel as shown in Table 1

37-Color Immunophenotyping Using Full-Spectrum Cytometry

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Fig. 4 Example of population selection for unmixing on single stain and unstained reference controls. Spectral plot (right) shows specific signature of each fluorochrome and cellular autofluorescence

demands a mention. Briefly, the most established method for cytometry data analysis is by a process known as “gating,” which uses a series of two-dimensional plots to identify regions of interest in a biaxial plot of single cells. A series of gates drawn in sequence can reveal information about cellular hierarchy and identify subsets from a population of interest (Figs. 5, 6, and

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Fig. 5 Manual Gating Strategy, part 1

37-Color Immunophenotyping Using Full-Spectrum Cytometry

Fig. 6 Manual Gating Strategy, part 2

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Fig. 7 Manual Gating Strategy, part 3

37-Color Immunophenotyping Using Full-Spectrum Cytometry

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Fig. 8 Visualization of high-dimensional data using t-SNE and UMAP algorithms on PBMC -excluding monocytes- from three concatenated samples. A. Biaxial plot with t-SNE (upper) and UMAP (lower) maps. B. Overlayed color-coded expression of several markers (CD3, CD4, CD8, TIGIT, CD57, CD19, IgM, IgD, CXCR5, CCR6, CD56, CD16 CD161, CD123 and CD27) on dimensional reduction t-SNE map. Data generated with the OMIQ Platform (https://omiq.ai)

7). Nevertheless, this is a subjective approach, labor-intensive and can potentially introduce bias. Analysis and visualization of data by dimensional reduction algorithms, relies in the identification of markers (dimensions or components), that explains the variation in a multi-dimensional dataset, due to biological causes, rather than technical artifacts [17, 18, 19]. Packages including tools for clustering and visualization of dimensional reduction analysis, are available either in R (FlowSOM; https://github.com/ SofieVG/FlowSOM), and Python (PhenoGraph; https:// github.com/jacoblevine/PhenoGraph; t-viSNE; https:// github.com/angeloschatzimparmpas/t-viSNE). Additional algorithms were integrated to pre-processing pipelines, to clean and normalize data before dimensional reduction and clustering are applied [20]. Dimensional reduction analysis of three samples, using our 37-color panel, is shown in Fig. 8

Acknowledgments This work was partially supported by Instituto de Salud Carlos III (Ministry of Health, Spain) grants COV20_00388, COV20_00416, and COV20_00654. Cytek Aurora Spectral Cytometer was funded by Spanish Ministry of Science and Innovation grant EQC2019-005914-P.

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References 1. Herzenberg LA, Sweet RG, Herzenberg LA (1976) Fluorescence-activated cell sorting. Sci Am 234(3):108–117. https://doi.org/10. 1038/scientificamerican0376-108 2. Biolegend history of flow cytometry. https:// www.biolegend.com/en-us/history-of-flow 3. Herzenberg LA, Parks D, Sahaf B et al (2002) The history and future of the fluorescence activated cell sorter and flow cytometry: a view from Stanford. Clin Chem 48:1819–1827. https://doi.org/10.1093/clinchem/48.10. 1819 4. Hu B, Guo H, Zhou P, Shi ZL (2021) Characteristics of SARS-CoV-2 and COVID-19. Nat Rev Microbiol 19(3):141–154. https:// doi.org/10.1038/s41579-020-00459-7 5. Nicola M, Alsafi Z, Sohrabi C et al (2020) The socio-economic implications of the coronavirus pandemic (COVID-19): a review. Int J Surg 78:185–193 6. John Hopkins Coronavirus Resource Center 7. Nolan JP, Condello D (2013) Spectral flow cytometry. Curr Protoc Cytom. https://doi. org/10.1002/0471142956.cy0127s63 8. Mahnke YD, Roederer M (2007) Optimizing a multicolor Immunophenotyping assay. Clin Lab Med 27(3):469–485, v 9. Hulspas R (2010) Titration of fluorochromeconjugated antibodies for labeling cell surface markers on live cells. Curr Protoc Cytom. https://doi.org/10.1002/0471142956. cy0629s54 10. Niewold P, Ashhurst TM, Smith AL, King NJC (2020) Evaluating spectral cytometry for immune profiling in viral disease. Cytometry A 97(11):1165–1179. https://doi.org/10. 1002/cyto.a.24211 11. Schmutz S, Valente M, Cumano A, Novault S (2016) Spectral cytometry has unique properties allowing multicolor analysis of cell suspensions isolated from solid tissues. PLoS One 11 (8):e0159961. https://doi.org/10.1371/jour nal.pone.0159961 12. Interim Laboratory biosafety guidelines for handling and processing specimens associated

with coronavirus disease 2019 (COVID-19). Centers Dis Control Prevention 13. Zhao Q, Meng M, Kumar R et al (2020) Lymphopenia is associated with severe coronavirus disease 2019 (COVID-19) infections: a systemic review and meta-analysis. Int J Infect Dis 96:131–135. https://doi.org/10.1016/j. ijid.2020.04.086 14. Jalbert E, Shikuma CM, Ndhlovu LC, Barbour JD (2013) Sequential staining improves detection of CCR2 and CX3CR1 on monocytes when simultaneously evaluating CCR5 by multicolor flow cytometry. Cytometry A 83 (3):280–286. https://doi.org/10.1002/cyto. a.22257 15. Ferrer-Font L, Pellefigues C, Mayer JU et al (2020) Panel design and optimization for highdimensional Immunophenotyping assays using spectral flow cytometry. Curr Protoc Cytom 92:1–25. https://doi.org/10.1002/cpcy.70 16. Park LM, Lannigan J, Jaimes MC (2020) Forty-color full spectrum flow cytometry panel for deep Immunophenotyping of major cell subsets in human peripheral blood. Cytometry A 97:1044–1051. https://doi.org/10. 1002/cyto.a.24213 17. Palit S, Heuser C, De Almeida GP et al (2019) Meeting the challenges of high-dimensional single-cell data analysis in immunology. Front Immunol 10:1515. https://doi.org/10. 3389/fimmu.2019.01515 18. Becht E, McInnes L, Healy J, et al (2019) Dimensionality reduction for visualizing single-cell data using UMAP. Nat Biotechnol https://doi.org/10.1038/nbt.4314 19. Amir EAD, Lee B, Badoual P et al (2019) Development of a comprehensive antibody staining database using a standardized analytics pipeline. Front Immunol 10:1315. https:// doi.org/10.3389/fimmu.2019.01315 20. Melsen JE, van Ostaijen-ten Dam MM, Lankester AC et al (2020) A comprehensive workflow for applying single-cell clustering and Pseudotime analysis to flow cytometry data. J Immunol 205(3):864–871. https://doi.org/ 10.4049/jimmunol.1901530

Chapter 5 Detection of Cytokine-Secreting Cells by Enzyme-Linked Immunospot (ELISpot) Bernt Axelsson Abstract The enzyme-linked immunospot (ELISpot) is a highly sensitive immunoassay that measures the frequency of cytokine-secreting cells at the single-cell level. The secreted molecules are detected by using a detection antibody system similar to that used in the enzyme-linked immunosorbent assay (ELISA). The ELISpot assay is carried out in a 96-well plate and an automated ELISpot reader is used for analysis. The assay is easy to perform, robust and allows rapid analysis of a large number of samples and is not limited to measurement of cytokines; it is suitable for almost any secreted protein where single-cell analysis is of interest. Key words 96-well plate, PVDF membrane, Cytokines/analytes, Capture antibodies, Detecting antibodies, Enzyme substrates

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Introduction The ELISpot method, originally developed to quantify immunoglobulin-secreting cells [1, 2], has evolved to be one of the most commonly used techniques to monitor immune responses by measuring the secretion of cytokines and other mediators from cells. It is used in a wide range of diseases, e.g., allergies [3, 4], infections [5–8], autoimmune diseases [9, 10], and cancer [11, 12]. The IFN-γ assay has been evaluated and validated in several proficiency panels [13, 14] and is considered a standard tool to evaluate antigen-specific T-cell immunity during the development of vaccine candidates and monitoring of new vaccines [6–8, 14–18]. In vaccine research and development, IFN-γ is often used as an immunocorrelate for CD8+ cytotoxic T-cell responses [7, 8], but other mediators such as granzyme B or perforin may be analyzed as well. A great advantage of the assay is the possibility to rapidly

Aik T. Ooi (ed.), Single-Cell Protein Analysis: Methods and Protocols, Methods in Molecular Biology, vol. 2386, https://doi.org/10.1007/978-1-0716-1771-7_5, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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screen a wide array of peptide antigens, allowing the detection of T-cell responses to a large pathogen using sets of overlapping peptides [9, 11, 16]. In the assay, cells are cultured in the presence or absence of stimuli on a membrane with an immobilized analyte-specific capture antibody. Proteins, such as cytokines, that are secreted by the activated cells will be captured by the specific antibodies on the surface. After an appropriate incubation time, the cells are washed away and an analyte-specific detection antibody is added. This antibody can be directly conjugated to an enzyme, but higher sensitivity is achieved if the antibody is biotinylated and followed by a streptavidin-enzyme conjugate. After a further incubation time, an enzyme substrate is added, a precipitating product is formed, and the end result is visible spots on the membrane (Fig. 1). Each spot corresponds to an individual cytokine-secreting cell.

ELISpot step-by-step guide B

1. Coating Monoclonal capture antibodies are added to

2. Cell incubation Cells are added in the presence of stimuli and the

3. Cytokine capture Secreted cytokines bind to capture antibodies

4. Detection antibodies Cells are removed and the plate is washed before

an ethanol-treated PVDF membrane plate

plate is incubated to allow cytokine secretion

surrounding the activated cells

biotinylated detection antibodies are added

ALP B

Biotin

SA

Streptavidin

SA

ALP

5. Streptavidin-enzyme

6. Addition of substrate

7. Analysis

conjugate Addition of a streptavidinconjugate enables the

A colorimetric substrate forms an insoluble precipitate when catalyzed

The result is analyzed in an automated spot reader. Each spot corresponds to a single

formation of spots on the membrane

by the enzyme

analyte-secreting cell

Alkaline phosphatase

Fig. 1 ELISpot step-by-step. The assay is a 3-d assay, including the preparation and coating of the plate on day 1 (1 h), loading of the plate with cells and stimuli on day 2 (4 h) and developing and reading of the plate on day 3 (6 h). Plates can be coated with the capture-antibody in advance and stored at 4
C for up to 2 weeks (reproduced with permission from Mabtech)

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The ELISpot assay captures the cytokine molecules immediately after they have been secreted and is considered as one of the most sensitive cellular assays available. The high sensitivity makes it useful for studies of specific immune responses where the responding cells constitutes only a small part of the population studied. The assay is robust and suitable for large-scale trials and for field studies. Plates precoated with capture antibodies and one-step detection reagents offer additional advantages. Although ELISpot measures the secretion of one cytokine per well it can be used to measure two cytokines in the same well [19]. This approach has, however, several limitations as it requires the use of two different enzymes and two substrates that generate different spot colors. Fluorospot, in which fluorescently labeled detection reagents are used, is a more accurate method for analyses of several analytes in the same well (see chapter on Fluorospot). B-cell ELISpot is one of a few assays that allows researchers to quantify cells secreting antibodies to a specific antigen as well as the total number of antibody-secreting cells in a cellular sample. By using specific detection antibodies, the isotype or subclass of secreted antibodies can be determined [1, 2]. With B-cell ELISpot it is also possible to demonstrate the presence and the frequencies of memory B cells in the blood [20, 21], which is difficult to assess by other methods. Today, diagnostic assays based on ELISpot are available, including a test to detect patients with tuberculosis infection by measuring IFN-γ secretion from T cells responding to defined antigens from Mycobacterium tuberculosis [22].

2

Materials Work under sterile conditions, preferably in a laminar flow hood, and use sterile materials when working with cells and cell culture medium. Follow the local regulations when working with human or animal cells and follow waste disposal regulations for work with such cells (see Note 1). In the present protocol, we have chosen IFN-γ secretion from stimulated PBMC to illustrate the cytokine detection process. ELISpot kits are available from several vendors and often include all necessary materials and a detailed instruction for performing the assay. Precoated plates offer additional benefits, such as shorter assay time, a reduced assay variation and a lower risk of handling errors.

2.1

Plate Coating

These materials are not required if precoated plates are used. 1. Plates with PVDF (polyvinylidene difluoride) membranes; MSIP plates, clear (MSIPS4510) or white format (MSIPS4W10) or Millipore MAIPSWU10 (Millipore, Cork, Ireland) (see Note 2).

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2. Ethanol in sterile dH2O (35% for MSIP plates and 70% for MAIPSWU10, freshly prepared from a stock of 99.5%) (see Note 3). 3. Sterile dH2O for washing of the plate (see Note 4). 4. Sterile container for wash water. 5. Multichannel micropipette (20–200 μL) and sterile, disposable pipette tips. 6. Container for waste disposal. 7. Sterile (autoclaved) PBS (phosphate-buffered saline) in, e.g., MilliQ water for dilution of antibody. 8. Sterile solution of cytokine-specific antibody, e.g., monoclonal anti-IFN-γ antibody (see Note 5). 2.2

Cell Culture

1. Sterile equipment for work with cells (5 mL and 10 mL pipettes, 50 mL and 15 mL centrifuge tubes, disposable pipette tips, containers, etc.) 2. Single and multichannel micropipettes, covering the range of 1–1000 μL. 3. Medium for cell culture and for conditioning of the plate wells (see Note 6). 4. Frozen or fresh cells, e.g., PBMC (see Note 7). 5. Equipment for cell counting, i.e., a Bu¨rker chamber for manual counting or an instrument for automated cell counting. 6. Trypan Blue solution (0.4%). 7. Table centrifuge intended for work with cells. 8. Sterile solutions of peptide antigens or other stimuli purchased from a vendor or manufactured in the laboratory (see Note 8). Follow the instructions from the vendor for the optimal concentrations to be used. 9. Positive control stimuli, e.g., Phytohemagglutinin (PHA) or anti-CD3 antibodies, used for assessment of cell viability and functionality of the assay (see Note 9). 10. Cell culture incubator with a humidified atmosphere (37
C and 5% CO2).

2.3 Detection and Analysis of Spots

1. Multi-pipette for manual washing or an automated plate washer adjusted to accommodate MSIP and MAIPSWU plates. 2. PBS for plate washing, diluted in, e.g., MilliQ water). 3. PBS containing 0.5% FCS for dilution of primary and secondary detection reagents (see Note 10). 4. Primary detection antibodies, e.g., biotin-labeled monoclonal anti-IFN-γ antibody directed to an epitope different from that of the capture antibody in Subheading 2.1 (see Note 11).

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5. Secondary reagents for amplification, i.e., Streptavidin-ALP or Streptavidin-HRP (see Note 11). 6. Ready-to-use precipitating enzyme substrates, e.g., BCIP/ NBT-Plus (for ALP) or TMB (for HRP) usually provided by the antibody supplier (see Note 12). 7. Tap water (for ALP) or deionized water (for HRP). 8. A dedicated spot reader equipped with a motorized stage, a digital camera, and an analysis software for counting of spots (see Note 13).

3

Methods Work under sterile conditions, preferably in a laminar flow hood, during Subheadings 3.1–3.4 (see Note 14). Avoid touching the membrane with the pipette-tips as this may cause leakage and/or staining artifacts in the well. It is very important not to let the well membranes in the plate dry out (become white) until the end of Subheading 3.5. To avoid staining artifacts, working reagent solutions should preferably be filtered (0.2 μm).

3.1 Pre-wetting of the Membrane and Addition of Capture Antibody

Steps 1–5 do not apply if precoated plates are used. 1. MSIP plates: add 15 μL of 35% ethanol to each well with a multichannel pipette. MAIPSWU plates: add 50 μL of 70% ethanol to each well. 2. After 30–60 s, start from the first row of added ethanol and fill each well with 200 μL of sterile MilliQ water using a multichannel pipette (see Note 15). 3. Empty the plate by inverting and flicking. 4. Wash the plate manually an additional 5 times with 200 μL/ well of sterile dH2O. Always remove the MAIPSWU10 plate from the plate tray before manually emptying the plate. Avoid liquid on the membrane backside and in the tray of MAIPSWU plates, as this can cause membrane leakage. Leave a small amount of PBS in each well to prevent membrane drying while diluting the capture antibodies. 5. Dilute the anti-IFN-γ capture antibody in sterile PBS to 10–15 μg/mL (follow the instructions from the vendor). 6. Empty the plate after the last wash and add 100 μL per well of the antibody solution. 7. Tap the side of the plate to avoid any air bubbles trapped in the wells. 8. Leave the plate over night at +4
C (see Note 16).

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3.2 Blocking/ Conditioning of the Plate Wells

1. Wash the plate 5 times with sterile PBS (200 μL/well), preferably with a multichannel pipette. 2. Add 200 μL/well of either PBS containing 10% FCS or complete cell culture medium. 3. Leave for 30 min (see Note 17).

3.3

Stimuli

Stimuli, e.g., antigen or other substances, can be added to the plate just before the addition of the cells, which is preferable for example if several stimuli are used or if stimuli need to be titrated. Do not titrate directly in the ELISpot plate. Use a titration plate and add the diluted stimuli from that plate to minimize the risk of damaging the well membranes. Cells and stimuli can also be mixed in vials and then added to the plate or cells can be pre-stimulated [23], preferably in round-bottom wells or tubes, to augment cell contact before added to the plate. 1. Prepare the working concentrations of the stimuli (2 the final concentrations) in complete medium. 2. Discard the blocking/conditioning solution from the plate. 3. Add antigen and control stimuli in 50 μL per well (see Note 18). 4. Include wells without stimuli (50 μL of complete medium) as controls for unstimulated cytokine release (preferably in triplicate or quadruplicate). 5. Include wells with medium only (no cells) for control of membrane background staining. 6. Gently tap the side of the plate to avoid air bubbles.

3.4

Addition of Cells

The protocol is based on the detection of specific IFN-γ responses by a heterogenous population of stimulated cells, e.g., PBMC. If other types of cells, e.g., antibody-secreting B cells (see Note 19), T-cell clones, and purified cell fractions, are under study, other protocols may have to be considered. If frozen cells are used, it is recommended that they are rested for at least 1 h, preferably overnight in a cell incubator to allow removal of cell debris before they are counted and added to the plate (see Note 7). 1. Count the cells manually by dilution in 0.4% Trypan Blue solution. Use a Bu¨rker chamber or a device for automated cell counting (see Note 20). 2. Adjust the cell concentration to 1–5  10E6 per mL of cell culture medium. 3. Suspend the cell suspension thoroughly before addition to the plate and ensure to avoid adding clumps of cells or remaining cell debris.

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4. Add 50.000–250.000 cells in 50 μL complete medium (see Note 21) to obtain a total volume of 100 μL in each well (see Subheading 3.3, step 1). 5. Carefully wrap the plate in aluminum foil to prevent evaporation while allowing gas exchange. 6. Gently tap the side of the plate a couple of times to distribute cells evenly. 7. Transfer the plate to the incubator. Ensure that the plate is flat on the surface and that the humidity of the incubator is satisfactory for small-volume cell cultures so that evaporation does not cause the wells to dry. Avoid sudden movements of the plate during cell incubation as this will negatively affect distinct spot formation. 8. Incubate cells overnight (this is sufficient for detection of IFN-γ released from stimulated PBMC) or longer depending on the kinetics of the cytokine tested. Do not move the plate during this time. 3.5 Detection of Spots (Footprint of Analyte-Secreting Cells)

Carry out all procedures at room temperature. Sterile conditions are not necessary.

3.5.1 Primary Reagent

1. After cell incubation, empty the plate by inverting and flicking (treat biological waste according to local regulations). 2. Wash 6 times with PBS using a manual pipette or an automated plate washer adapted to the ELISpot plate. Leave a small amount of wash buffer in each well while diluting the detection reagents (see Note 22). 3. Dilute biotinylated anti-IFN-γ detection antibody in PBS with 0.5% FCS according to instructions from the antibody provider (usually about 1 μg/mL) (see Note 23). 4. Empty the plate of residual wash buffer by inverting and flicking and add 100 μL per well of diluted antibody. 5. Tap the side of the plate to avoid air bubbles and leave the plate for 1.5–2 h at room temperature.

3.5.2 Secondary Reagent

1. Wash as in Subheading 3.5.1, step 2. 2. Dilute Streptavidin-ALP or Streptavidin-HRP in PBS with 0.5% FCS according to the provider of the reagent (usually 1/1000) (see Note 24). 3. Empty the plate of residual wash buffer by inverting and flicking and add 100 μL per well of the diluted reagent.

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4. Tap the side of the plate to avoid air bubbles and leave the plate for 1 h at room temperature. 5. Wash as in Subheading 3.5.1, step 2. 6. Filter (0.45 μm) the ready-to-use enzyme substrate buffer (BCIP/NBT-Plus for ALP and TMB for HRP). 7. Empty the plate by inverting and flicking and add 100 μL per well of the substrate. 8. Follow the reaction under a dissection microscope or other magnifying device to be able to interrupt when distinct spots emerge, usually after 2–15 min (see Note 25). A general darkening of the membrane may occur but usually disappears after drying. 9. Stop color development by washing Streptavidin-ALP plates in tap water several times; rinse plates with TMB substrate in deionized water. 10. Leave the plate to dry, preferably upside-down in a laminar flow hood (about 1 h) with the plastic underlid removed. 11. Store the plate at room temperature in the dark until analysis. 3.6 Analysis of the Plate

1. Analyze the dry plate in a dedicated ELISpot reader (see Note 13) (Fig. 2). 2. Calculate the frequency of responding cells by determining the mean number of stimulated spot-forming cells (duplicates or triplicates) by subtracting the mean number of spot-forming cells in unstimulated wells (triplicates or quadruplicates). Usually, the threshold defining a significant response is set to exceed three standard deviations of the number of spotforming cells in unstimulated wells (negative control) (see Note 26). 3. Check the result in the plate to see if troubleshooting is needed when any of the following is observed. (a) No or few detectable spots seen (see Note 27). (b) Spots are blurry (see Note 28). (c) Staining artifacts are seen in the well (see Note 29). (d) The membrane background is nonspecifically stained (see Note 30). (e) TMB spots show color variation or fade (see Note 31). (f) A positive response in negative control wells (see Note 32). (g) Spot numbers do not correlate with cell numbers (show linearity) in the assay (see Note 33).

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Fig. 2 ELISpot analysis of PBMC stimulated by antigen (CEF peptide pool) or stimulated polyclonally (anti-CD3 antibody). Black spots represent detection of IFN-γ-secreting cells with Streptavidin-ALP using BCIP/NBT-Plus as substrate and blue spots represent detection with Streptavidin-HRP and TMB as substrate (reproduced with permission from Mabtech)

4

Notes 1. Good microbiological practice is essential in ELISpot procedures. An aseptic technique should be used in order to minimize microbiological contamination of the cells and the plate wells and to protect the user from any potential hazards from the cells. Biological material, including blood, cells, serum, plasma, and supernatants should be treated as potentially infected. 2. ELISpot 96-well plates are available from several manufacturers, but the most commonly used are from Merck-Millipore. It is recommended to use plates equipped with PVDF (polyvinylidene difluoride) membranes which have a high proteinbinding capacity. Plates with nitrocellulose membranes can also be used but may yield less consistent results. MSIP plates from Merck-Millipore have a removable plastic underdrain attached to the bottom surface and are available in clear (MSIPS4510) or white format (MSIPS4W10). The two formats give a slightly different appearance in the ELISpot reader, but this will not affect the results. MAIPSWU10 plates, also called ELIIP, come in a white format. Instead of an underdrain, the plate is placed in a detachable tray.

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3. Maximum protein-binding capacity of PVDF membranes is obtained by a brief treatment with ethanol (diluted from stock 99.5%). 4. Autoclaved pure water, e.g., from a MilliQ apparatus. The sterile solutions should be handled as such, to be opened as few times as possible and only under aseptic conditions in sterile hoods. Do not use solutions older than 3 months for sterile purposes. 5. Follow the instructions from the antibody supplier. We use a stock solution of anti-human mAb 1-D1K at 1 mg/mL (Mabtech AB, Nacka Strand, Sweden) diluted to a working concentration of 15 μg/mL in sterile PBS. 6. For human peripheral blood mononuclear cells (PBMC) and mouse spleen cells, RPMI 1640 (+ Glutamax) supplemented with 10% fetal calf serum (FCS), HEPES (10 mM), and a mix of penicillin ( IFN-γ/IL-22 > IFN-γ/IL-17A (reproduced with permission from Mabtech AB)

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2. Calculate the frequency of responding cells secreting a certain cytokine or a combination of cytokines in stimulated wells (duplicates or triplicates) by subtracting the number of spotforming cells secreting the same cytokine or combination of cytokines in unstimulated wells (triplicates or quadruplicates). Usually, the threshold defining a significant response is set to exceed three standard deviations of the number of spotforming cells in unstimulated wells (negative control) (see Note 26). 3. Check the results in the plate to see if troubleshooting is needed when any of the following is observed. (a) No or few detectable spots seen (see Note 27). (b) Blurry spots (see Note 28). (c) Staining artifacts are seen in the well (see Note 29). (d) The membrane background is nonspecifically stained (see Note 30). (e) A positive response in negative control wells (see Note 31), (f) Cytokine absorption effects (see Note 32). (g) Spot numbers do not correlate with cell numbers (show linearity) in the assay (see Note 33).

4

Notes 1. Good microbiological practice is essential in FluoroSpot procedures. An aseptic technique should be used in order to minimize microbiological contamination of the cells and the plate wells and to protect the user from any potential hazards from the cells. Biological material, including blood, cells, serum, plasma, and supernatants should be treated as potentially infected. 2. Transparent plates with polyvinylidene difluoride (PVDF) membranes with low autofluorescence (IPFL) are available from Merck-Millipore. Apart from the membrane, the IPFL plate has the same configuration and characteristics as the transparent MSIPS4510 plate from Millipore. IPFL plates have a removable plastic underdrain attached to the bottom surface. White plates should be avoided due to the impact of interfering light reflections on plate analysis. 3. Maximum protein binding capacity of PVDF membranes is obtained by a brief treatment with ethanol (diluted from stock 99.5%). 4. Autoclaved pure water, e.g., from a MilliQ apparatus. The sterile solutions should be handled as such, to be opened as

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few times as possible and only under aseptic conditions in sterile hoods. Do not use solutions older than 3 months for sterile purposes. 5. Follow the instructions from the antibody supplier. Using 15μg/mL of each capture antibody and 100μL per well, each well membrane will be coated with about 6μg of antibody if four analytes are assayed. This is well within the binding capacity (roughly 100μg per well) of an ethanol-treated PVDF well membrane. 6. RPMI 1640 (+Glutamax) supplemented with 10% fetal calf serum (FCS), HEPES (10 mM), and a mix of penicillin (