OMIP-053
Identification, Classification, and Isolation of Major FoxP3 Expressing Human CD4+ Treg Subsets
Cytometry PART A, Volume 95, Issue 3, 264-267 (2019)
Johannes Nowatzky, Cristy Stagnar, Olivier Manches
Description: 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.
Species: Human
Tissue: Cell line, Peripheral blood
Cell Subset: T cell, T memory, Treg
Keyword: Activation, Autoimmunity, Cryopreserved pbmc, Proliferation
Machine: BD LSR2
Panel details
| Marker | Species | Clone | Fluorophore | Purpose | Co-expressions groups | Antigen Density |
|---|---|---|---|---|---|---|
| Dead cells | Human | Live/Dead Fix Blue | Viability | |||
| CD3 | Human | SK7 | PerCP-Cy5.5 | Lineage | T cell, T memory, Treg | High |
| CD127 | Human | A019D5 | Brilliant Violet 421 | Treg (TDP I) | T cell, Treg | Low |
| CD4 | Human | SK3 | Brilliant Violet 510 | Lineage | T cell, Treg | High |
| CD45RA | Human | HI100 | Brilliant Violet 711 | Treg (TDP II), Treg activation, | T cell, T memory | Med |
| CD25 | Human | M-A251 | PE-Dazzle 594 | Treg (TDP I and II) | T cell, Treg | Low |
| CD38 | Human | HB7 | Brilliant Ultraviolet 395 | Activation (BTREG) | T cell, Treg, T memory | Low |
| CD39 | Human | A1 | APC-Fire 750 | suppressive capcity and stability under inflammatory condition (BTREG) | T cell, Treg, T memory | Low |
| FOXP3 | Human | 236A/E7 | APC | Treg (TDP I -III) (ATREG) | T cell, Treg | Low |
| Ki-67 | Human | B56 | Brilliant Ultraviolet 395 | Proliferation (ATREG) | T cell | Low |
| Helios | Human | 22F6 | PE-Cy7 | Treg (TDP III) (ATREG) | T cell, Treg | Med |
| CD8a | Human | SK1 | Brilliant Violet 650 | CD8 exclusion | T cell, T memory | High |
| HLA DR | Human | L243 | FITC | Activation | T cell, T memory | High |
| CCR7 | Human | G043H7 | PE | Naïve/memory classification | T cell, T memory | Low |
| TIGIT | Human | MBSA43 | APC | Treg identification | T cell, Treg | Low |
| CD226 | Human | 11A8 | PE-Cy7 | exclusion of functionally unstable Treg for in vitro expansion, Treg identification (BTREG) | T cell, Treg | Med |
