Note: In order to make the guidelines as beneficial as possible to the scientific community, if you wish to refer to a specific section when referring to these guidelines, please ensure to use the Chapter and Section information. For example: …as noted in section VII.11 of [1] … 1. Cossarizza, A., Chang, H. D., Radbruch, A., Acs, A., Adam, A., Adam-Klages, S., Agace, W. et al., Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition). Eur. J. Immunol. 2019. 49: 1457–1973.The final version of the second edition published by WIley-VCH is freely available online at: https://doi.org/10.1002/eji.201970107 The third edition was published online at https://doi.org/10.1002/eji.202170126 on 7 December 2021 under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits use, distribution and reproduction in any medium, provided the original work is properly cited.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.Work in the laboratory of Dieter Adam is supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)—Projektnummer 125440785 – SFB 877, Project B2.
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.
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.
Jessica G Borger, Kylie M Quinn, Mairi McGrath, and Regina Stark thank Francesco Siracusa and Patrick Maschmeyer for providing data.
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).
Susanne Hartmann and Friederike Ebner were supported by the German Research Foundation (GRK 2046).
Hans Minderman was supported by NIH R50CA211108.
This work was funded by the Deutsche Forschungsgemeinschaft through the grant TRR130 (project P11 and C03) to Thomas H. Winkler.
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).
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).
Axel Ronald Schulz, Antonio Cosma, Sabine Baumgart, Brice Gaudilliere, Helen M. McGuire, and Henrik E. Mei thank Michael D. Leipold for critically reading the manuscript.
Christian Kukat acknowledges support from the ISAC SRL Emerging Leaders program.
John Trowsdale received funding from the European Research Council under the European Union's Horizon 2020 research and innovation program (Grant Agreement 695551)
