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

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.

Yolk sac erythromyeloid progenitors sustain erythropoiesis throughout embryonic life

“…436Stained cells were analyzed on a custom BD LSR Fortessa or BD FACSymphony and 437 were sorted with a BD FACSAria III (BD Biosciences) according to the guidelines for 438 the use of flow cytometry and cell sorting(Cossarizza et al, 2019). Data were analyzed 439 with FlowJo software (v.10.5.3, BD Biosciences) or R packages as described in 440 "Bioinformatic Analysis".…”

MiSet RFC Standards: Defining a Universal Minimum Set of Standards Required for Reproducibility and Rigor in Research Flow Cytometry Experiments

“…Minimum standards have also been defined in the setting of flow cytometry SRLs, providing guidance for best practices in instrumentation, training, experimental design, biosafety standards, and sample handling (11,20,21). For the specific needs of the immunology community, a comprehensive guide covering the principles, techniques and applications of immunology flow cytometry has been published by the European Journal of Immunology (22). These substantial efforts emphasize the need for standard definitions to create robust and reproducible results in the setting of specialized experimental settings, research questions, sample types, and access to instrumentation or the expertise of SRLs/flow cytometry collaborators.…”

“…The International Society for the Advancement of Cytometry (ISAC) is an expert flow cytometry community that provides consensus recommendations to accelerate flow cytometry research in the setting of cross-disciplinary collaboration. ISAC endorses several resources that help to define, annotate, and report flow cytometry experiments (e.g., optimized multicolor immunofluorescence panels (OMIPs) (18) and MIFlowCyt (19)), as well as recommendations issued for specific settings of immunology research and SRLs (11,(20)(21)(22). However, these resources have not found widespread adaption, and very few scientific journals and publishers currently require adherence to MIFlowCyt standards or deposition of experimental data and methodology.…”

Horizontal gene transfer in the human and skin commensal Malassezia: a bacterially-derived flavohemoglobin is required for NO resistance and host interaction

“…Cells were acquired on a FACS Cytoflex (Beckman Coulter) and the data were analyzed with FlowJo software (FlowJo LLC). The gating of the flow cytometric data was performed according to the guidelines for the use of flow cytometry and cell sorting in immunological studies 85 , including pre-gating on viable and single cells for analysis. For transcript expression analysis, total RNA was isolated from ear skin according to standard protocols using TRI Reagent (Sigma Aldrich).…”