Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition)

Andrea Cossarizza, Hyun Dong Chang, Andreas Radbruch, Andreas Acs, Dieter Adam, Sabine Adam-Klages, William W. Agace, Nima Aghaeepour, Mübeccel Akdis, Matthieu Allez, Larissa Nogueira Almeida, Giorgia Alvisi, Graham Anderson, Immanuel Andrä, Francesco Annunziato, Achille Anselmo, Petra Bacher, Cosima T. Baldari, Sudipto Bari, Vincenzo BarnabaJoana Barros-Martins, Luca Battistini, Wolfgang Bauer, Sabine Baumgart, Nicole Baumgarth, Dirk Baumjohann, Bianka Baying, Mary Bebawy, Burkhard Becher, Wolfgang Beisker, Vladimir Benes, Rudi Beyaert, Alfonso Blanco, Dominic A. Boardman, Christian Bogdan, Jessica G. Borger, Giovanna Borsellino, Philip E. Boulais, Jolene A. Bradford, Dirk Brenner, Ryan R. Brinkman, Anna E.S. Brooks, Dirk H. Busch, Martin Büscher, Timothy P. Bushnell, Federica Calzetti, Garth Cameron, Ilenia Cammarata, Xuetao Cao, Susanna L. Cardell, Stefano Casola, Marco A. Cassatella, Andrea Cavani, Antonio Celada, Lucienne Chatenoud, Pratip K. Chattopadhyay, Sue Chow, Eleni Christakou, Luka Čičin-Šain, Mario Clerici, Federico S. Colombo, Laura Cook, Anne Cooke, Andrea M. Cooper, Alexandra J. Corbett, Antonio Cosma, Lorenzo Cosmi, Pierre G. Coulie, Ana Cumano, Ljiljana Cvetkovic, Van Duc Dang, Chantip Dang-Heine, Martin S. Davey, Derek Davies, Sara De Biasi, Genny Del Zotto, Gelo Victoriano Dela Cruz, Michael Delacher, Silvia Della Bella, Paolo Dellabona, Günnur Deniz, Mark Dessing, James P. Di Santo, Andreas Diefenbach, Francesco Dieli, Andreas Dolf, Thomas Dörner, Regine J. Dress, Diana Dudziak, Michael Dustin, Charles Antoine Dutertre, Friederike Ebner, Sidonia B.G. Eckle, Matthias Edinger, Pascale Eede, Götz R.A. Ehrhardt, Marcus Eich, Pablo Engel, Britta Engelhardt, Anna Erdei, Charlotte Esser, Bart Everts, Maximilien Evrard, Christine S. Falk, Todd A. Fehniger, Mar Felipo-Benavent, Helen Ferry, Markus Feuerer, Andrew Filby, Kata Filkor, Simon Fillatreau, Marie Follo, Irmgard Förster, John Foster, Gemma A. Foulds, Britta Frehse, Paul S. Frenette, Stefan Frischbutter, Wolfgang Fritzsche, David W. Galbraith, Anastasia Gangaev, Natalio Garbi, Brice Gaudilliere, Ricardo T. Gazzinelli, Jens Geginat, Wilhelm Gerner, Nicholas A. Gherardin, Kamran Ghoreschi, Lara Gibellini, Florent Ginhoux, Keisuke Goda, Dale I. Godfrey, Christoph Goettlinger, Jose M. González-Navajas, Carl S. Goodyear, Andrea Gori, Jane L. Grogan, Daryl Grummitt, Andreas Grützkau, Claudia Haftmann, Jonas Hahn, Hamida Hammad, Günter Hämmerling, Leo Hansmann, Goran Hansson, Christopher M. Harpur, Susanne Hartmann, Andrea Hauser, Anja E. Hauser, David L. Haviland, David Hedley, Daniela C. Hernández, Guadalupe Herrera, Martin Herrmann, Christoph Hess, Thomas Höfer, Petra Hoffmann, Kristin Hogquist, Tristan Holland, Thomas Höllt, Rikard Holmdahl, Pleun Hombrink, Jessica P. Houston, Bimba F. Hoyer, Bo Huang, Fang Ping Huang, Johanna E. Huber, Jochen Huehn, Michael Hundemer, Christopher A. Hunter, William Y.K. Hwang, Anna Iannone, Florian Ingelfinger, Sabine M. Ivison, Hans Martin Jäck, Peter K. Jani, Beatriz Jávega, Stipan Jonjic, Toralf Kaiser, Tomas Kalina, Thomas Kamradt, Stefan H.E. Kaufmann, Baerbel Keller, Steven L.C. Ketelaars, Ahad Khalilnezhad, Srijit Khan, Jan Kisielow, Paul Klenerman, Jasmin Knopf, Hui Fern Koay, Katja Kobow, Jay K. Kolls, Wan Ting Kong, Manfred Kopf, Thomas Korn, Katharina Kriegsmann, Hendy Kristyanto, Thomas Kroneis, Andreas Krueger, Jenny Kühne, Christian Kukat, Désirée Kunkel, Heike Kunze-Schumacher, Tomohiro Kurosaki, Christian Kurts, Pia Kvistborg, Immanuel Kwok, Jonathan Landry, Olivier Lantz, Paola Lanuti, Francesca LaRosa, Agnès Lehuen, Salomé LeibundGut-Landmann, Michael D. Leipold, Leslie Y.T. Leung, Megan K. Levings, Andreia C. Lino, Francesco Liotta, Virginia Litwin, Yanling Liu, Hans Gustaf Ljunggren, Michael Lohoff, Giovanna Lombardi, Lilly Lopez, Miguel López-Botet, Amy E. Lovett-Racke, Erik Lubberts, Herve Luche, Burkhard Ludewig, Enrico Lugli, Sebastian Lunemann, Holden T. Maecker, Laura Maggi, Orla Maguire, Florian Mair, Kerstin H. Mair, Alberto Mantovani, Rudolf A. Manz, Aaron J. Marshall, Alicia Martínez-Romero, Glòria Martrus, Ivana Marventano, Wlodzimierz Maslinski, Giuseppe Matarese, Anna Vittoria Mattioli, Christian Maueröder, Alessio Mazzoni, James McCluskey, Mairi McGrath, Helen M. McGuire, Iain B. McInnes, Henrik E. Mei, Fritz Melchers, Susanne Melzer, Dirk Mielenz, Stephen D. Miller, Kingston H.G. Mills, Hans Minderman, Jenny Mjösberg, Jonni Moore, Barry Moran, Lorenzo Moretta, Tim R. Mosmann, Susann Müller, Gabriele Multhoff, Luis Enrique Muñoz, Christian Münz, Toshinori Nakayama, Milena Nasi, Katrin Neumann, Lai Guan Ng, Antonia Niedobitek, Sussan Nourshargh, Gabriel Núñez, José Enrique O'Connor, Aaron Ochel, Anna Oja, Diana Ordonez, Alberto Orfao, Eva Orlowski-Oliver, Wenjun Ouyang, Annette Oxenius, Raghavendra Palankar, Isabel Panse, Kovit Pattanapanyasat, Malte Paulsen, Dinko Pavlinic, Livius Penter, Pärt Peterson, Christian Peth, Jordi Petriz, Federica Piancone, Winfried F. Pickl, Silvia Piconese, Marcello Pinti, A. Graham Pockley, Malgorzata Justyna Podolska, Zhiyong Poon, Katharina Pracht, Immo Prinz, Carlo E.M. Pucillo, Sally A. Quataert, Linda Quatrini, Kylie M. Quinn, Helena Radbruch, Tim R.D.J. Radstake, Susann Rahmig, Hans Peter Rahn, Bartek Rajwa, Gevitha Ravichandran, Yotam Raz, Jonathan A. Rebhahn, Diether Recktenwald, Dorothea Reimer, Caetano Reis e Sousa, Ester B.M. Remmerswaal, Lisa Richter, Laura G. Rico, Andy Riddell, Aja M. Rieger, J. Paul Robinson, Chiara Romagnani, Anna Rubartelli, Jürgen Ruland, Armin Saalmüller, Yvan Saeys, Takashi Saito, Shimon Sakaguchi, Francisco Sala-de-Oyanguren, Yvonne Samstag, Sharon Sanderson, Inga Sandrock, Angela Santoni, Ramon Bellmàs Sanz, Marina Saresella, Catherine Sautes-Fridman, Birgit Sawitzki, Linda Schadt, Alexander Scheffold, Hans U. Scherer, Matthias Schiemann, Frank A. Schildberg, Esther Schimisky, Andreas Schlitzer, Josephine Schlosser, Stephan Schmid, Steffen Schmitt, Kilian Schober, Daniel Schraivogel, Wolfgang Schuh, Thomas Schüler, Reiner Schulte, Axel Ronald Schulz, Sebastian R. Schulz, Cristiano Scottá, Daniel Scott-Algara, David P. Sester, T. Vincent Shankey, Bruno Silva-Santos, Anna Katharina Simon, Katarzyna M. Sitnik, Silvano Sozzani, Daniel E. Speiser, Josef Spidlen, Anders Stahlberg, Alan M. Stall, Natalie Stanley, Regina Stark, Christina Stehle, Tobit Steinmetz, Hannes Stockinger, Yousuke Takahama, Kiyoshi Takeda, Leonard Tan, Attila Tárnok, Gisa Tiegs, Gergely Toldi, Julia Tornack, Elisabetta Traggiai, Mohamed Trebak, Timothy I.M. Tree, Joe Trotter, John Trowsdale, Maria Tsoumakidou, Henning Ulrich, Sophia Urbanczyk, Willem van de Veen, Maries van den Broek, Edwin van der Pol, Sofie Van Gassen, Gert Van Isterdael, René A.W. van Lier, Marc Veldhoen, Salvador Vento-Asturias, Paulo Vieira, David Voehringer, Hans Dieter Volk, Anouk von Borstel, Konrad von Volkmann, Ari Waisman, Rachael V. Walker, Paul K. Wallace, Sa A. Wang, Xin M. Wang, Michael D. Ward, Kirsten A. Ward-Hartstonge, Klaus Warnatz, Gary Warnes, Sarah Warth, Claudia Waskow, James V. Watson, Carsten Watzl, Leonie Wegener, Thomas Weisenburger, Annika Wiedemann, Jürgen Wienands, Anneke Wilharm, Robert John Wilkinson, Gerald Willimsky, James B. Wing, Rieke Winkelmann, Thomas H. Winkler, Oliver F. Wirz, Alicia Wong, Peter Wurst, Jennie H.M. Yang, Juhao Yang, Maria Yazdanbakhsh, Liping Yu, Alice Yue, Hanlin Zhang, Yi Zhao, Susanne Maria Ziegler, Christina Zielinski, Jakob Zimmermann, Arturo Zychlinsky

Research output: Contribution to journalArticlepeer-review

206 Scopus citations

Abstract

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.

Original languageEnglish (US)
Pages (from-to)1457-1973
Number of pages517
JournalEuropean Journal of Immunology
Volume49
Issue number10
DOIs
StatePublished - Oct 1 2019

Bibliographical note

Funding Information:
Ramon Bellmàs Sanz, Jenny Kühne, and Christine S. Falk thank Jana Keil and Kerstin Daemen for excellent technical support. The work was funded by the Germany Research Foundation CRC738/B3 (CSF).

Funding Information:
Larissa Nogueira Almeida was supported by DFG research grant MA 2273/14‐1. Rudolf A. Manz was supported by the Excellence Cluster “Inflammation at Interfaces” (EXC 306/2).

Funding Information:
Susanne Hartmann and Friederike Ebner were supported by the German Research Foundation (GRK 2046).

Funding Information:
Edwin van der Pol and Paola Lanuti acknowledge Aleksandra Gąsecka M.D. for excellent experimental support and Dr. Rienk Nieuwland for textual suggestions. This work was supported by the Netherlands Organisation for Scientific Research – Domain Applied and Engineering Sciences (NWO‐TTW), research program VENI 15924.

Funding Information:
Hans Minderman was supported by NIH R50CA211108.

Funding Information:
Work in the laboratory of Dieter Adam is supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)—Projektnummer 125440785 – SFB 877, Project B2.

Funding Information:
Christian Kukat acknowledges support from the ISAC SRL Emerging Leaders program.

Funding Information:
This work was funded by the Deutsche Forschungsgemeinschaft through the grant TRR130 (project P11 and C03) to Thomas H. Winkler.

Funding Information:
The Immunology Database and Analysis Portal (ImmPort) system provides an archive of immunology research data generated by investigators mainly funded through the National Institutes of Health (NIH), National Institute of Allergy and Infectious Diseases (NIAID), and Division of Allergy, Immunology, and Transplantation (DAIT). It is an extensive data warehouse containing an integration of experimental and clinical trial data generated by dozens of assay types, including 131 FCM and 24 CyTOF data sets. In addition, the ImmPort system also provides data analysis tools and it contains implicit knowledge and ‘‘best practices’’ for clinical and genomic studies in the form of ∼50 templates for data deposition, management, and dissemination. ImmPort has been developed under the Bioinformatics Integration Support Contract (BISC) by the Northrop Grumman Information Technology Health Solutions team for the NIH NIAID/DAIT. If your research funding comes from this source and you are generating immunology data, you should deposit it in ImmPort. Immport's support for different data types can be another reason to choose it if you are generating FCM data as well as data from different types of assays. A (free) registration and approval by DAIT is required in order to deposit and access data from ImmPort. A potential drawback of using this resource is that there can be a long delay in publication of deposited datasets.

Funding Information:
The work by the Mei laboratory was supported by German Research Foundation Grant ME 3644/5‐1 and TRR130 TP24, the German Rheumatism Research Centre Berlin, European Union Innovative Medicines Initiative ‐ Joint Undertaking ‐ RTCure Grant Agreement 777357, the Else Kröner‐Fresenius‐Foundation, German Federal Ministry of Education and Research e:Med sysINFLAME Program Grant 01ZX1306B and KMU‐innovativ “InnoCyt”, and the Leibniz Science Campus for Chronic Inflammation ( http://www.chronische-entzuendung.org ).

Funding Information:
Petra Hoffmann, Andrea Hauser, and Matthias Edinger thank BD Biosciences®, San José, CA, USA, and SKAN AG, Bale, Switzerland for fruitful cooperation during the development, construction, and installation of the GMP‐compliant cell sorting equipment and the Bavarian Immune Therapy Network (BayImmuNet) for financial support.

Funding Information:
John Trowsdale received funding from the European Research Council under the European Union's Horizon 2020 research and innovation program (Grant Agreement 695551).

Publisher Copyright:
© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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