Table Of Content

1584 Andrea Cossarizza et al. DOI: 10.1002/eji.201646632 Eur. J. Immunol. 2017. 47: 1584–1797 HIGHLIGHTS Guidelines for the use of flow cytometry and cell sorting in immunological studies ∗1 ∗2 ∗2 193 Andrea Cossarizza , Hyun-Dong Chang , Andreas Radbruch , Mu¨beccel Akdis , 3 4 5 6,7 9 Immanuel Andra¨ , Francesco Annunziato , Petra Bacher , Vincenzo Barnaba , Luca Battistini , 10 2 11 12 2 Wolfgang M. Bauer , Sabine Baumgart , Burkhard Becher , Wolfgang Beisker , Claudia Berek , 13 9 14,15 16,17 Alfonso Blanco , Giovanna Borsellino , Philip E. Boulais , Ryan R. Brinkman , 18 3,19,20 21 22,23,24 25 Martin Bu¨scher , Dirk H. Busch , Timothy P. Bushnell , Xuetao Cao , Andrea Cavani , 26 27 28 29 30 Pratip K. Chattopadhyay , Qingyu Cheng , Sue Chow , Mario Clerici , Anne Cooke , 31 32 35 2 36 Antonio Cosma , Lorenzo Cosmi , Ana Cumano , Van Duc Dang , Derek Davies , Sara De 33 37 38,39 40 41 Biasi , Genny Del Zotto , Silvia Della Bella , Paolo Dellabona , Gu¨nnur Deniz , 42 5 43 44 45 Mark Dessing , Andreas Diefenbach , James Di Santo , Francesco Dieli , Andreas Dolf , 46 47 48 49 50 Vera S. Donnenberg , Thomas Do¨rner , Go¨tz R. A. Ehrhardt , Elmar Endl , Pablo Engel , 51 52 53 193 54,55 Britta Engelhardt , Charlotte Esser , Bart Everts , Anita Dreher , Christine S. Falk , 56 57 58,59,60 61 62 Todd A. Fehniger , Andrew Filby , Simon Fillatreau , Marie Follo , Irmgard Fo¨rster , 63 64 14,65 66 45,67 John Foster , Gemma A. Foulds , Paul S. Frenette , David Galbraith , Natalio Garbi , 68 194 69 33 Maria Dolores Garc´ıa-Godoy , Jens Geginat , Kamran Ghoreschi , Lara Gibellini , 70 71 72 73 74 Christoph Goettlinger , Carl S. Goodyear , Andrea Gori , Jane Grogan , Mor Gross , 2 63 75 2 2,76 Andreas Gru¨tzkau , Daryl Grummitt , Jonas Hahn , Quirin Hammer , Anja E. Hauser , 77 28 78 75 27 David L. Haviland , David Hedley , Guadalupe Herrera , Martin Herrmann , Falk Hiepe , 67 79 80 27 81,82,83 Tristan Holland , Pleun Hombrink , Jessica P. Houston , Bimba F. Hoyer , Bo Huang , 84 85 86 87 88,89 Christopher A. Hunter , Anna Iannone , Hans-Martin Ja¨ck , Beatriz Ja´vega , Stipan Jonjic , 2 74 2 90 91 48 Kerstin Juelke , Steffen Jung , Toralf Kaiser , Tomas Kalina , Baerbel Keller , Srijit Khan , 75 92 93 45 94 Deborah Kienho¨fer , Thomas Kroneis , Désirée Kunkel , Christian Kurts , Pia Kvistborg , 95 96,97,98 99,100,101,102 103 Joanne Lannigan , Olivier Lantz , Anis Larbi , Salome LeibundGut-Landmann , 104 105 106 48 107 Michael D. Leipold , Megan K. Levings , Virginia Litwin , Yanling Liu , Michael Lohoff , 108 109 110 111 Giovanna Lombardi , Lilly Lopez , Amy Lovett-Racke , Erik Lubberts , 112 113,114 104 115 Burkhard Ludewig , Enrico Lugli , Holden T. Maecker , Glo`ria Martrus , 116 75 2 71 2 Giuseppe Matarese , Christian Mauero¨der , Mairi McGrath , Iain McInnes , Henrik E. Mei , 117 118 119 8 193 Fritz Melchers , Susanne Melzer , Dirk Mielenz , Kingston Mills , David Mirrer , 120,121 122 8 123,124 Jenny Mjo¨sberg , Jonni Moore , Barry Moran , Alessandro Moretta , 125 126 127 128 11 Lorenzo Moretta , Tim R. Mosmann , Susann Mu¨ller , Werner Mu¨ller , Christian Mu¨nz , 129,130 75 131,132 Gabriele Multhoff , Luis Enrique Munoz , Kenneth M. Murphy , 133 33 54 134 Toshinori Nakayama , Milena Nasi , Christine Neudo¨rfl , John Nolan , 135 87 136 137 Sussan Nourshargh , José-Enrique O’Connor , Wenjun Ouyang , Annette Oxenius , 138 139 140 18 68 Raghav Palankar , Isabel Panse , Pa¨rt Peterson , Christian Peth , Jordi Petriz , 94 141 6,7 34 64,142 Daisy Philips , Winfried Pickl , Silvia Piconese , Marcello Pinti , A. Graham Pockley , Correspondence: Prof. Andrea Cossarizza and Dr. Hyun-Dong Chan e-mail: Eur. J. Immunol. 2017. 47: 1584–1797 Guidelines for the use of flow cytometry and cell sorting in immunological studies 1585 75 143 126 Malgorzata Justyna Podolska , Carlo Pucillo , Sally A. Quataert , 144 145 126 146 Timothy R. D. J. Radstake , Bartek Rajwa , Jonathan A. Rebhahn , Diether Recktenwald , 147 148 68 149 Ester B.M. Remmerswaal , Katy Rezvani , Laura G. Rico , J. Paul Robinson , 2 150 193 151,152,153 Chiara Romagnani , Anna Rubartelli , Beate Ruckert , Ju¨rgen Ruland , 154,155 87 156 Shimon Sakaguchi , Francisco Sala-de-Oyanguren , Yvonne Samstag , 157 158,159 5,2 3 Sharon Sanderson , Birgit Sawitzki , Alexander Scheffold , Matthias Schiemann , 160 161 162 163 Frank Schildberg , Esther Schimisky , Stephan A Schmid , Steffen Schmitt , 3 164 2 94 108 Kilian Schober , Thomas Schu¨ler , Axel Ronald Schulz , Ton Schumacher , Cristiano Scotta , 165 74 139 16 166 T. Vincent Shankey , Anat Shemer , Anna-Katharina Simon , Josef Spidlen , Alan M. Stall , 79 2 119 119 167 Regina Stark , Christina Stehle , Merle Stein , Tobit Steinmetz , Hannes Stockinger , 168 169,170 171,172 173 Yousuke Takahama , Attila Tarnok , ZhiGang Tian , Gergely Toldi , 117 174 166 175 Julia Tornack , Elisabetta Traggiai , Joe Trotter , Henning Ulrich , Marlous van der 94 79 176 67 177 Braber , René A. W. van Lier , Marc Veldhoen , Salvador Vento-Asturias , Paulo Vieira , 178 179 180 181 David Voehringer , Hans-Dieter Volk , Konrad von Volkmann , Ari Waisman , 182 183 91 93 184 Rachael Walker , Michael D. Ward , Klaus Warnatz , Sarah Warth , James V. Watson , 185 18 47 186 Carsten Watzl , Leonie Wegener , Annika Wiedemann , Ju¨rgen Wienands , 187 154,155 45 188 189 Gerald Willimsky , James Wing , Peter Wurst , Liping Yu , Alice Yue , 190 191 115 192 Qianjun Zhang , Yi Zhao , Susanne Ziegler and Jakob Zimmermann 1 Department of Medical and Surgical Sciences for Children and Adults, Univ. of Modena and Reggio Emilia School of Medicine, Modena, Italy 2 Deutsches Rheuma-Forschungszentrum (DRFZ), an Institute of the Leibniz Association, Berlin, Germany 3 Institut fu¨r Medizinische Mikrobiologie, Immunologie und Hygiene, Technische Universita¨t Mu¨nchen, Munich, Germany 4 University of Florence, Internal Medicine, Florence, Italy 5 Charité - Universita¨tsmedizin Berlin, Germany 6 Dipartimento di Medicina Interna e Specialita` Mediche, Sapienza Universita` di Roma, Via Regina Elena 324, 00161 Rome, Italy 7 Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Rome, Italy 8 Trinity Biomedical Sciences Institute, Trinity College Dublin, the University of Dublin, Dublin, Ireland 9 Neuroimmunology and Flow Cytometry Units, Santa Lucia Foundation, Rome, Italy 10 Division of Immunology, Allergy and Infectious Diseases, Department of Dermatology, Medical University of Vienna, Vienna, Austria 11 University of Zurich, Institute of Experimental Immunology, Zu¨rich, Switzerland 12 Flow Cytometry Laboratory, Institute of Molecular Toxicology and Pharmacology, Helmholtz Zentrum Mu¨nchen, German Research Center for Environmental Health 13 Flow Cytometry Core Technologies, UCD Conway Institute, University College Dublin, Dublin, Ireland 14 Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York, USA 15 The Ruth L. and David S. Gottesman Institute for Stem Cell and Regenerative Medicine Research, Bronx, New York, USA 16 Terry Fox Laboratory, BC Cancer Agency, Vancouver, BC, Canada 17 Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada 18 Biopyhsics, R&D Engineering, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany 19 DZIF - National Centre for Infection Research, Munich, Germany 20 Focus Group ‘‘Clinical Cell Processing and Purification”, Institute for Advanced Study, Technische Universita¨t Mu¨nchen, Munich, Germany 21 Department of Pediatrics and Shared Resource Laboratories, University of Rochester Medical Center, Rochester NY, United States of America 22 Institute of Immunology, Zhejiang University School of Medicine, Hangzhou 310058, China 23 National Key Laboratory of Medical Immunology & Institute of Immunology, Second Military Medical University, Shanghai 200433, China 24 Department of Immunology & Center for Immunotherapy, Institute of Basic Medical Sciences, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing 100005, China ⃝C 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu 1586 Andrea Cossarizza et al. Eur. J. Immunol. 2017. 47: 1584–1797 25 INMP/NIHMP, Rome, Italy 26 Immunotechnology Section, Vaccine Research Center, NIH, Bethesda, MD, USA 27 Medizinische Klinik mit Schwerpunkt Rheumatologie und Medizinische Immunolologie Charité Universita¨tsmedizin Berlin, Berlin, Germany 28 Divsion of Medical Oncology and Hematology, Princess Margaret Hospital, Toronto, Ontario, Canada 29 University of Milano and Don C Gnocchi Foundation IRCCS, Milano, Italy 30 Department of Pathology, University of Cambridge, Cambridge, United Kingdom 31 CEA - Université Paris Sud - INSERM U, Immunology of viral infections and autoimmune diseases, France 32 Department of Experimental and Clinical Medicine, University of Firenze, Firenze, Italia 33 Department of Surgery, Medicine, Dentistry and Morphological Sciences, Univ. of Modena and Reggio Emilia, Modena, Italy 34 Department of Life Sciences, Univ. of Modena and Reggio Emilia, Modena, Italy 35 Lymphopoiesis Unit, Immunology Department Pasteur Institute, Paris, France 36 Flow Cytometry Facility, The Francis Crick Institute, London, United Kingdom 37 Istituto Giannina Gaslini, Genova, Italy 38 University of Milan, Department of Medical Biotechnologies and Translational Medicine 39 Humanitas Clinical and Research Center, Lab of Clinical and Experimental Immunology, Rozzano, Milan, Italy 40 Experimental Immunology Unit, Head, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milano, Italy 41 Istanbul University, Aziz Sancar Institute of Experimental Medicine, Department of Immunology, Istanbul, Turkey 42 Sony Europe Ltd, Weybridge, UK 43 Institut Pasteur, Immunology, Paris, France 44 University of Palermo, Department of Biopathology, Palermo, Italy 45 Institute of Experimental Immunology, University Bonn, Bonn, Germany 46 Department of Cardiothoracic Surgery, School of Medicine, University of Pittsburgh, PA 47 Department of Medicine/Rheumatology and Clinical Immunology, Charite Universita¨tsmedizin Berlin, Germany 48 Department of Immunology, University of Toronto, Toronto, Canada 49 Department of Molecular Medicine and Experimental Immunology, (Core Facility Flow Cytometry) University of Bonn, Germany 50 Department of Biomedical Sciences, University of Barcelona, Barcelona, Spain 51 Professor for Immunobiology, Director, Theodor Kocher Institute, University of Bern, Bern, Switzerland 52 IUF – Leibniz Research Institute for Environmental Medicine, Du¨sseldorf, Germany 53 Leiden University Medical Center, Department of Parasitology, Leiden, The Netherlands 54 Institute of Transplant Immunology, IFB-Tx, MHH Hannover Medical School, Hannover, Germany 55 German Center for Infectious diseases (DZIF), TTU-IICH, Hannover, Germany 56 Divisions of Hematology & Oncology, Department of Medicine, Washington University School of Medicine, St Louis, MO 57 The Flow Cytometry Core Facility, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, UK 58 Institut Necker-Enfants Malades (INEM), INSERM U-CNRS UMR, Paris, France 59 Université Paris Descartes, Sorbonne Paris Cité, Faculté de Médecine, Paris, France 60 Assistance Publique - Hoˆpitaux de Paris (AP-HP), Hoˆpital Necker Enfants Malades, Paris, France 61 Department of Medicine I, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany 62 Immunology and Environment, Life & Medical Sciences (LIMES) Institute, University of Bonn, Bonn, Germany 63 Owl Biomedical Inc., Santa Barbara, USA 64 John van Geest Cancer Research Centre, Nottingham Trent University, Nottingham, UK 65 Department of Medicine, Albert Einstein College of Medicine, Bronx, New York, USA 66 University of Arizona, Bio Institute, School of Plant Sciences and Arizona Cancer Center, Tucson, Arizona, USA 67 Department of Molecular Immunology, Institute of Experimental Immunology, Bonn, Germany 68 Josep Carreras Leukemia Research Institute, Barcelona, Spain 69 Flow Cytometry Core Facility, Department of Dermatology, University Medical Center, Eberhard Karls University Tu¨bingen, Germany 70 Institut fuer Genetik, Universitaet zu Koeln, Koeln, Germany 71 Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow 72 Clinic of Infectious Diseases, "San Gerardo” Hospital - ASST Monza, University Milano-Bicocca, Monza, Italy 73 Genentech, Department of Cancer Immunology, South San Francisco, California, USA 74 Department of Immunology, Weizmann Institute of Science, Rehovot, Israel ⃝C 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu Eur. J. Immunol. 2017. 47: 1584–1797 Guidelines for the use of flow cytometry and cell sorting in immunological studies 1587 75 Friedrich-Alexander-University Erlangen-Nu¨rnberg (FAU), Department of Internal Medicine, Rheumatology and Immunology, Universita¨tsklinikum Erlangen, Erlangen 76 Immundynamics, Charité - Universita¨tsmedizin Berlin, Berlin, Germany 77 Houston Methodist Hospital Research Institute, Houston, TX, USA 78 Cytometry Service, Incliva Foundation. Clinic Hospital and Faculty of Medicine, The University of Valencia. Av. Blasco Ibań˜ez, Valencia, Spain 79 Department of Hematopoiesis, Sanquin Research and Landsteiner Laboratory, Amsterdam, The Netherlands 80 Chemical and Materials Engineering, New Mexico State University, Las Cruces, NM, 88003, USA 81 Department of Biochemistry and Molecular Biology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China 82 Department of Immunology, Institute of Basic Medical Sciences & State Key Laboratory of Medical Molecular Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China 83 Clinical Immunology Center, Chinese Academy of Medical Sciences, Beijing, China 84 Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA 85 Department of Diagnostic Medicine, Clinical and Public Health, Univ. of Modena and Reggio Emilia, Modena, Italy 86 Division of Molecular Immunology, Internal Medicine III, Nikolaus-Fiebiger-Center of MolecularMedicine, University Hospital Erlangen, Erlangen, Germany 87 Laboratory of Cytomics, Joint Research Unit CIPF-UVEG, Department of Biochemistry and Molecular Biology, The University of Valencia. Av. Blasco Ibań˜ez, Valencia, Spain 88 Faculty of Medicine, Center for Proteomics, University of Rijeka, Rijeka, Croatia 89 Department for Histology and Embryology, Faculty of Medicine, University of Rijeka, Rijeka, Croatia 90 Department of Paediatric Haematology and Oncology, Second Faculty of Medicine, Charles University and University Hospital Motol, Prague, Czech Republic 91 Center for Chronic Immunodeficiency (CCI), Faculty of Medicine, University of Freiburg, Freiburg, Germany 92 Medical University of Graz, Institute of Cell Biology, Histology & Embryology, Graz, Austria 93 BCRT Flow Cytometry Lab, Berlin-Brandenburg Center for Regenerative Therapies, Charité - Universita¨tsmedizin Berlin 94 Division of immunology, the Netherlands Cancer Institute, Amsterdam 95 University of Virginia School of Medicine, Flow Cytometry Shared Resource, Charlottesville, VA, USA 96 INSERM U932, Institut Curie, Paris 75005, France 97 Laboratoire d’immunologie clinique, Institut Curie, Paris 75005, France 98 Centre d’investigation Clinique en Biothérapie Gustave-Roussy Institut Curie (CIC-BT1428), Institut Curie, Paris 75005, France 99 Singapore Immunology Network (SIgN), Principal Investigator, Biology of Aging Program 100 Director Flow Cytomerty Platform, Immunomonitoring Platform, Agency for Science Technology and Research (A*STAR), Singapore 101 Department of Medicine, University of Sherbrooke, Qc, Canada 102 Faculty of Sciences, ElManar University, Tunis, Tunisia 103 Section of Immunology, Vetsuisse Faculty, University of Zurich, Switzerland 104 The Human Immune Monitoring Center (HIMC), Institute for Immunity, Transplantation and Infection, Stanford University School of Medicine, CA, USA 105 Department of Surgery, University of British Columbia & British Columbia Children’s Hospital Research Institute, Vancouver, BC, Canada 106 Covance, Hematology, Indianapolis, IN, USA 107 Institute for Medical Microbiology and Hospital Hygiene, University of Marburg, Marburg 35043, Germany 108 MRC Centre for Transplantation, King’s College London, Guy’s Hospital, SE1 9RT London, UK 109 Beckman Coulter, Inc, Miami, FL, USA 110 Department of Microbial Infection and Immunity, Ohio State University, Columbus, OH, USA 111 Erasmus MC, University Medical Center, Department of Rheumatology, Rotterdam, The Netherlands 112 Institute of Immunobiology, Kantonsspital St. Gallen, St. Gallen, Switzerland 113 Laboratory of Translational Immunology, Humanitas Clinical and Research Center, Rozzano, Milan, Italy 114 Humanitas Flow Cytometry Core, Humanitas Clinical and Research Center, Rozzano, Milan, Italy 115 Department of Virus Immunology, Heinrich-Pette-Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany 116 Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Universita` di Napoli Federico II, Napoli, Italy and Istituto per l’Endocrinologia e l’Oncologia Sperimentale, Consiglio Nazionale delle Ricerche (IEOS-CNR), Napoli, Italy 117 Senior Group on Lymphocyte Development, Max Planck Institute for Infection Biology, Berlin, Germany ⃝C 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu 1588 Andrea Cossarizza et al. Eur. J. Immunol. 2017. 47: 1584–1797 118 Clinical Trial Center Leipzig, University Leipzig, Leipzig, Germany 119 Division of Molecular Immunology, Nikolaus-Fiebiger-Center, Dept. of Internal Medicine III, University of Erlangen-Nuremberg, Erlangen, Germany 120 Center for Infectious Medicine, Department of Medicine, Karolinska Institute Stockholm, Sweden 121 Department of Clinical and Experimental Medicine, Linko¨ping University, Sweden 122 Department of Pathology and Laboratory Medicine, Perelman School of Medicine of the University of Pennsylvania, Philadelphia, Pennsylvania 123 Department of Experimental Medicine, University of Genova, Genova, Italy 124 Centro di Eccellenza per la Ricerca Biomedica—CEBR, Genova, Italy 125 Department of Immunology, IRCCS Bambino Gesu Children’s Hospital, Rome, Italy 126 David H. Smith Center for Vaccine Biology and Immunology, University of Rochester Medical Center, Rochester, NY, USA 127 Centre for Environmental Research - UFZ, Department Environemntal Microbiology, Leipzig, Germany 128 Bill Ford Chair in Cellular Immunology, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, United Kingdom 129 Department of Radiation Oncology, Klinikum rechts der Isar, Technische Universita¨t Mu¨nchen (TUM), Munich, Germany 130 Institute for Innovative Radiotherapy (iRT), Experimental Immune Biology, Helmholtz Zentrum Mu¨nchen, Neuherberg, Germany 131 Department of Pathology and Immunology, School of Medicine, Washington University in St. Louis, St. Louis, MO, USA 132 Howard Hughes Medical Institute, School of Medicine, Washington University in St. Louis, St. Louis, MO, USA 133 Department of Immunology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260–8670, Japan 134 The Scintillon Institute, Nancy Ridge Drive, San Diego, CA, USA 135 Centre for Microvascular Research, William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom 136 Department of Inflammation and Oncology, Amgen Inc., South San Francisco, CA, USA 137 Institute of Microbiology, ETH Zurich, Zurich, Switzerland 138 Institute for Immunology and Transfusion Medicine, University Medicine Greifswald, Ferdinand-Sauerbruch-Straße, 17489, Greifswald, Germany 139 Kennedy Institute of Rheumatology, University of Oxford, Oxford, United Kingdom 140 Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia 141 Institute of Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria 142 Chromocyte Limited, Electric Works, Sheffield, UK 143 Univeristy of Udine - Department of Medicine, Lab of Immunology, Udine, Italy 144 Department of Rheumatology and Clinical Immunology, University Medical Center Utrecht, Utrecht, The Netherlands; Laboratory of Translational Immunology, University Medical Center Utrecht, Utrecht, The Netherlands 145 Bindley Biosciences Center, Purdue University, West Lafayette, In, USA 146 Desatoya LLC, Reno, NV, USA 147 Department of Experimental Immunology and Renal Transplant Unit, Division of Internal Medicine, Academic Medical Centre, The Netherlands 148 Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA 149 The SVM Professor of Cytomics & Professor of Biomedical Engineering, Purdue University Cytometry Laboratories, Purdue University, West Lafayette, IN, USA 150 Cell Biology Unit, IRCCS AOU San Martino-IST, Genova, Italy 151 Institut fu¨r Klinische Chemie und Pathobiochemie, Klinikum rechts der Isar, Technische Universita¨t Mu¨nchen, Munich, Germany 152 German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany 153 German Center for Infection Research (DZIF), partner site Munich, Munich, Germany 154 Laboratory of Experimental Immunology, WPI Immunology Frontier Research Center (IFReC), Osaka University, Suita 565–0871, Japan 155 Department of Experimental Pathology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606–8507, Japan 156 Institute of Immunology, Section Molecular Immunology, Ruprecht-Karls-University, D-69120, Heidelberg, Germany 157 Translational Immunology Laboratory, NIHR BRC, University of Oxford, Kennedy Institute of Rheumatology,Oxford, United Kingdom ⃝C 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu Eur. J. Immunol. 2017. 47: 1584–1797 Guidelines for the use of flow cytometry and cell sorting in immunological studies 1589 158 Charité-Universitaetsmedizin Berlin, Corporate Member of Freie Universitaet Berlin, Humboldt-Universitaet zu Berlin 159 Berlin Institute of Health, Institute of Medical Immunology, Augustenburger Platz 1, 13353 Berlin, Germany 160 Harvard Medical School, Department of Microbiology and Immunobiology, Boston, MA, USA 161 R&D Reagents, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany 162 Klinik und Poliklinik fu¨r Innere Medizin I, Universita¨tsklinikum Regensburg, Regensburg, Germany 163 Imaging and Cytometry Core Facility, Flow Cytometry Unit, German Cancer Research Centre (DKFZ), Heidelberg, Germany 164 Institute of Molecular and Clinical Immunology, Otto-von-Guericke University, Magdeburg, Germany 165 AsedaSciences, West Lafayette, IN, USA 166 BD Life Sciences, San Diego, CA USA 167 Institute for Hygiene and Applied Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria 168 Division of Experimental Immunology, Institute of Advanced Medical Sciences, University of Tokushima, Tokushima, Japan 169 Departement for Therapy Validation, Fraunhofer Institute for Cell Therapy and Immunology IZI, Leipzig, Germany 170 Institute for Medical Informatics, IMISE, Leipzig, Germany 171 School of Life Sciences and Medical Center, Institute of Immunology, Key Laboratory of Innate Immunity and Chronic Disease of Chinese Academy of Science, University of Science and Technology of China, Hefei, China 172 Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China 173 University of Birmingham, Institute of Immunology and Immunotherapy, Birmingham, UK 174 Novartis Institute for Biomedical Research (NIBR), Basel Switzerland 175 Departamento de Bioqu´ımica, Instituto de Qu´ımica, Universidade de Saõ Paulo 176 Instituto de Medicina Molecular, Lisbon, Portugal 177 Unité Lymphopoiese, Institut Pasteur, Paris, France 178 Department of Infection Biology, University Hospital Erlangen, Wasserturmstr. 3/5, 91054 Erlangen, Germany 179 Inst. Med. Immunol. & BCRT & Labor Berlin 180 A·P·E-Applied Physics & Electronics, Berlin, Germany 181 Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg University of Mainz, Mainz, Germany 182 Babraham Institute, Cambridge, UK 183 Thermo Fisher Scientific, Eugene, Oregon, USA 184 Medinfomatics Ltd., West Drayton, Middlesex, England 185 Leibniz Research Centre for Working Environment and Human Factors at TU Dortmund, IfADo, Department of Immunology, Dortmund, Germany 186 Universita¨tsmedizin Go¨ttingen, Georg-August-Universita¨t, Abt. Zellula¨re und Molekulare Immunologie, Humboldtallee 34, 37073 Go¨ttingen, Germany 187 Cooperation Unit for Experimental and Translational Cancer Immunology, Institute of Immunology (Charité - Universita¨tsmedizin Berlin) and German Cancer Research Center (DKFZ), Berlin, Germany 188 BD Biosciences, San Jose CA, USA 189 School of Computing Science, Simon Fraser University, Burnaby, Canada 190 Emerald Biotech Co., Ltd, Hangzhou, China 191 Department of Rheumatology & Immunology, West China Hospital, Sichuan University, Chengdu, China 192 Maurice Mu¨ller Laboratories (DKF), Universita¨tsklinik fu¨r Viszerale Chirurgie und Medizin Inselspital, University of Bern, Murtenstrasse, Bern 193 Swiss Institute of Allergy and Asthma Research (SIAF), University Zurich, Davos, Switzerland 194 INGM, Istituto Nazionale Genetica Molecolare “Romeo ed Enrica Invernizzi”, Milan, Italy ⃝C 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu 1590 Andrea Cossarizza et al. Eur. J. Immunol. 2017. 47: 1584–1797 Contents Introduction: Guidelines for the use of flow cytometry in immunology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1595 I. Cytometry equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1596 1. Fluidic system of a flow cytometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1596 1.1 Purposes of the fluidic system of a flow cytometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1596 1.2 Hydrodynamic focusing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1596 1.3 Acoustic focusing of particles in a liquid stream .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1597 1.4 Droplet generation of a cell sorter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1597 2. Optics and electronics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1598 2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1598 2.2 Optics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1598 2.3 Electronics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1599 3. Flow cytometry, including flow cytometry cell sorting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1600 3.1 Convention, or fluorescence-activated flow cytometry and sorting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1600 3.2 Spectral flow cytometry: Principles and evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1601 4. Imaging flow cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1602 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1602 4.2 Imaging flow cytometers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1603 4.3 Experimental set-up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1603 4.4 Acquisition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1604 4.5 Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1604 5. Mass cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1604 5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1604 5.2 Mass cytometry in biological research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1605 5.3 The mass cytometer: Cell introduction and signal detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1605 5.4 Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1606 5.5 Bottlenecks in mass cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1606 5.6 Experimental workflow, reagents, and controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1607 5.7 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1608 II. Cell sorting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1608 Introduction to cell sorting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1608 1. Parallel cell sorting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1609 1.1 Introduction and general considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1609 1.2 Antibody based bulk cell sorting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1610 1.3 Methods based on density differences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1611 1.4 Methods based on cell size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1612 1.5 Erythrocyte lysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1613 1.6 A historical note . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1613 2. Serial cell sorting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1613 2.1 Cell sorting by flow cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1613 2.2 Microfluidic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1616 III. Setup: Instrument setup and quality control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1618 1. Compensation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1618 1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1618 1.2 Principle of spillover and compensation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1618 1.3 Measuring SOVs/compensation controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1618 1.4 Compensation controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1620 Stained cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1620 Beads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1620 1.5 What are “good” SOVs? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1620 1.6 What is “good enough” accuracy for SOVs? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1620 2 Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1621 2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1621 2.2 Cleaning of instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1621 ⃝C 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu Eur. J. Immunol. 2017. 47: 1584–1797 Guidelines for the use of flow cytometry and cell sorting in immunological studies 1591 2.3 Computer and software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1625 IV. Before you start: Reagent and sample preparation, experimental design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1625 1 Controls: Determining positivity by eliminating false positives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1625 1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1625 1.2 Fluorescence spreading into the channel of interest: FMO controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1625 1.3 Specificity of reagent for staining target: Biological controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1626 1.4 Specificity of reagent for staining target: Blocking and isotype controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1626 2 Titration: Determining optimal reagent concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1626 3 Preparation of single-cell suspensions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1627 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1627 3.2 Mechanical disintegration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1628 3.3 Enzymatic digestion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1628 3.4 Special disaggregation techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1628 3.5 Ensuring a single-cell suspension/removing oversized aggregates after extraction . . . . . . . . . . . . . . . . . . . . . . . . . . 1628 3.6 General comments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1629 4 Pre-enrichment of low abundant cell populations prior to acquisition/cell sorting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1629 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1629 4.2 Pre-enrichment by physical properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1629 4.3 Pre-enrichment by immunological properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1630 5 Frozen samples and cell viability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1631 5.1 Freezing cell samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1631 5.2 Testing for cell viability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1632 6 Cell fixation and permeabilization for flow cytometric analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1634 6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1634 6.2 Fixation of whole blood specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1634 6.3 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1635 6.4 Effect of methanol on epitope staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1636 6.5 Fixation and permeabilization for non-adherent tissue culture cell preparations . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1637 7 Barcoding in cytometric assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1638 8 Key concepts for the design and testing of multicolor panels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1642 9 Variable lymphocyte receptor antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1643 9.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1643 9.2 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1644 9.3 Two-layer staining approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1644 9.4 Three-layer staining approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1644 V. Data acquisition and cell sorting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1645 1 Suspended sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1645 1.1 Nozzle diameter, clogging, and cell filtration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1645 1.2 How to keep cells in suspension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1645 2 Trigger, thresholds, and live gating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1646 2.1 Trigger and threshold . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1646 2.2 Trigger parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1647 2.3 Live gating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1647 2.4 Threshold and cell sorting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1647 3 Rare cells: General rules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1647 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1647 3.2 Optimization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1647 3.3 Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1650 4 Collecting cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1650 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1650 4.2 Cell sorter-specific parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1650 4.3 Sample collection tubes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1651 VI. Evaluation and data handling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1651 1 Data analysis: An overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1651 1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1651 1.2 Pre-processing flow data in preparation for sub-population identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1651 ⃝C 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu 1592 Andrea Cossarizza et al. Eur. J. Immunol. 2017. 47: 1584–1797 1.3 Identification of sub-population sizes and properties by gating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1653 1.4 Post-processing of sub-population data: Comparison of experimental groups and identification of significantly altered sub-populations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1654 2 Data analysis—automated analysis: Automated flow cytometry cell population identification and visualization . . . . . . . 1654 2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1654 2.2 Visualizing big flow cytometry data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1655 2.3 Next steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1655 3 Statistics for flow cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1655 3.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1655 3.2 Probability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1656 3.3 Types of distributions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1656 3.4 Distribution parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1657 3.5 Significance testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1657 3.6 An example of immunofluorescent staining in cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1660 3.7 Rare cell analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1662 4 Analysis presentation and publication (MIFlowCyt) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1663 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1663 4.2 Minimal display requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1663 4.3 Presentation checklist for publication (MIFlowCyt) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1665 5 Data repositories: Sharing your data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1666 VII. Cytometric parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1668 1 Organisms, cells, organelles, and chromosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1668 1.1 Organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1668 1.2 Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1668 1.3 Nuclei . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1668 1.4 Cell organelles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1668 1.5 Chromosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1669 2 Surface parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1669 2.1 Minimize artefacts by minimal cell manipulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1669 2.2 Exclude dead cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1670 2.3 Magnetic pre-enrichment for high-resolution detection and analysis of rare cell populations . . . . . . . . . . . . . . 1670 2.4 Transient surface markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1670 2.5 Genuine membrane molecules versus membrane adsorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1671 2.6 Quantitative considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1671 3 Intracellular parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1671 4 Combinatorial cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1673 5 Measuring antigen specific T-cell responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1678 5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1678 5.2 UV light-mediated peptide exchange method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1678 5.3 Staining and flow cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1678 5.4 Example: Detection of neo-antigen specific T-cell responses in a melanoma patient . . . . . . . . . . . . . . . . . . . . . . . . . 1679 6 Antigen-specific T-cell cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1680 6.1 MHC multimers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1680 6.2 Functional read-outs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1682 7 DNA synthesis, cell cycle, and proliferation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1686 7.1 DNA synthesis and cell cycle analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1687 7.2 Proliferation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1689 7.3 Useful resources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1690 8 Cell death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1691 8.1 DNA-binding dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1691 8.2 Protein-binding dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1692 8.3 Vital dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1692 8.4 Measurement of apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1693 8.5 Caspase activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1694 8.6 Cytofluorimetric analysis of mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1695 ⃝C 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu Eur. J. Immunol. 2017. 47: 1584–1797 Guidelines for the use of flow cytometry and cell sorting in immunological studies 1593 9 Phagocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1698 9.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1698 9.2 Flow cytometric assays of phagocytosis: fundamentals and general applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1699 9.3 Critical points in the pre-analytical and analytical phases of assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1699 10 Autophagy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1702 10.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1702 10.2 Flow cytometry autophagy assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1702 10.3 Measuring autophagy in primary cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1703 11 Cytotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1704 12 Reactive oxygen species production with minimal sample perturbation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1706 12.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1706 12.2 Sample preparation and flow cytometry setup for measuring ROS generation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1707 2+ 13 Intracellular Ca mobilization by means of Indo-1 AM .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1709 13.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1709 2+ 13.2 Theory of measuring intracellular Ca mobilization v

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