Standardized 11-color flow cytometry panel for the functional phenotyping of human T regulatory cells


  • Claire Manuszak Dana-Farber Cancer Institute Center for Immuno-Oncology
  • Martha Brainard Dana-Farber Cancer Institute Center for Immuno-Oncology
  • Emily Thrash Dana-Farber Cancer Institute Center for Immuno-Oncology
  • F. Stephen Hodi Dana-Farber Cancer Institute Center for Immuno-Oncology
  • Mariano Severgnini Dana-Farber Cancer Institute Center for Immuno-Oncology



Tregs, flow cytometry, antibody panel, peripheral blood mononuclear cells


T regulatory cells (Tregs) are a cell subset that can suppress immune responses to maintain homeostasis and self-tolerance. In some scenarios, the immunosuppressive nature could be associated to other pathological developments such as autoimmune diseases and cancers. Due to the importance of Tregs in disease pathogenesis, we developed and validated an 11-color flow cytometry panel for phenotypic and functional detection of Treg markers using healthy human donor peripheral blood mononuclear cells (PBMCs). Our panel contains 4 Treg surface proteins and 2 functional cytokines as well as T-lymphocyte lineage markers CD3, CD4, and CD8. Our data shows an increase in expression of markers CD25, FoxP3, CTLA4, GITR and intracellular cytokines IL4 and TGFβ when comparing unstimulated samples to CD3/CD28 bead stimulated samples. This 11-color panel can be used to functionally evaluate immunosuppressive Tregs in human PBMC samples.

Author Biography

Mariano Severgnini, Dana-Farber Cancer Institute Center for Immuno-Oncology

I am the Lead Scientist and Director of the Center for Immuno-Oncology Immune Assessment Laboratory


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How to Cite

Manuszak C, Brainard M, Thrash E, Hodi FS, Severgnini M. Standardized 11-color flow cytometry panel for the functional phenotyping of human T regulatory cells. J Biol Methods [Internet]. 2020Apr.13 [cited 2022Aug.11];7(2):e131. Available from:




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