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Identification, Classification, and Isolation of Major FoxP3 Expressing Human CD4+ Treg Subsets

Johannes Nowatzky, Cristy Stagnar, Olivier Manches Cytometry PART A, Volume 95, Issue 3, 264-267 (2019)

PURPOSE: We designed and optimized an inter‐convertible flow cytometry panel for the analysis and sorting of human CD4+ regulatory T cells (Treg) utilizing all of the major, currently accepted marker combinations for Treg identification. The panel is optimized for use with peripheral blood mononuclear cells (PBMC). In addition, the panel allows for the identification, classification, and isolation of activated, antigen‐experienced Treg, and provides estimates of proliferation and suppressive capacity. To this end we created two, easily inter‐convertible sub‐panels: ATREG—which includes intra‐nuclear markers, and BTREG—using surface markers only. Both sub‐panels have been tested on cryopreserved PBMC from healthy donors and those with human autoimmune diseases, as well as on long‐term cultured human Treg and non‐Treg cell lines and clones.

CELL TYPE: PBMC, long‐term cultured Treg


Marker Target Clone Fluorochrome Purpose
Dead cells Human Live/Dead Fix Blue Viability
HLA-DR Human L243 FITC Activation
CD3 Human SK7 PerCP-Cy5.5 Lineage
TIGIT Human MBSA43 APC Treg identification
CD127 Human A019D5 Brilliant Violet 421 Treg (TDP I)
CD4 Human SK3 Brilliant Violet 510 Lineage
CD8 Human SK1 Brilliant Violet 650 CD8 exclusion
CD45RA Human HI100 Brilliant Violet 711 Treg (TDP II), Treg activation,
CD25 Human M-A251 PE-Dazzle 594 Treg (TDP I and II)
ccr7 Human G043H7 PE Naïve/memory classification
CD38 Human HB7 BUV395 Activation (BTREG)
CD226 Human 11A8 PE-Cy7 exclusion of functionally unstable Treg for in vitro expansion, Treg identification (BTREG)
CD39 Human A1 APC-Fire 750 suppressive capcity and stability under inflammatory condition (BTREG)
FoxP3 Human 236A/E7 APC Treg (TDP I -III) (ATREG)
Ki-67 Human B56 BUV395 Proliferation (ATREG)
Helios Human 22F6 PE-Cy7 Treg (TDP III) (ATREG)