OMIPs Directory

OMIP-060

30‐Parameter Flow Cytometry Panel to Assess T Cell Effector Functions and Regulatory T Cells

Cytometry PART A, Volume 95, Issue 11, 1129-1134 (2019)
Thomas Liechti, Mario Roederer

Description: We developed this comprehensive 28‐color flow cytometry panel with the aim to measure a variety of T cell effector functions in combination with T cell differentiation markers (CCR7, CD27, CD28, CD45RO, CD95) in γδ T cells and CD4+ and CD8+ αβ T cells. The effector functions measured in this panel include activation and co‐stimulatory molecules (CD69, CD137, and CD154), cytokines (IL‐2, IL‐13, IL‐17A, IL‐21, IL‐22, TNF, and IFNγ), the chemokine IL‐8, cytotoxic molecules (perforin and granzyme B), and the degranulation marker CD107a. In addition, Ki67 enables the identification and analysis of recently activated T cells. To characterize regulatory T cells (Tregs), we included CD25, CD39, and the canonical Tregs transcription factor FoxP3. We developed and optimized this panel for cryopreserved human peripheral blood mononuclear cells (PBMC) and stimulation with phorbol 12‐myristate 13‐acetate (PMA) and ionomycin. However, we successfully tested other types of stimulation such as staphylococcus enterotoxin B (SEB) or a mix of immunodominant peptides (CEF peptide pool) from cytomegalovirus (CMV), Epstein–Barr virus (EBV) and influenza.

Species: Human

Tissue: Peripheral blood

Cell Subset: T cell, Ctl, T helper, T memory, Tcrgd, Th1, Th17, Th22

Keyword: Antigen-specific t cell response, Cytomegalovirus (cmv), Epstein-barr virus (ebv), Immune checkpoint, Influenza, Pma/ ionomycin, Seb

Machine: BD FACSymphony A5

Panel details

Marker Species Clone Fluorophore Purpose Co-expressions groups Antigen Density
IL-8 Human G265-8 Brilliant Blue 630-P2 Effector function T cell, T helper, CTL Low
IL-13 Human JES10-5A2 Brilliant Blue 660-P2 Effector function T cell, T helper Low
CD137 Human 4B4-1 Brilliant Blue 790-P Co-stimulatory T cell, T memory, T helper, Treg Low
TCR gamma/delta Human B1 PE-Cy5 γδ T cell γδ T cell Low
Dead cells Human Live/Dead Fix Blue viability
CD28 Human CD28.2 PE Differentiation T cell, γδ T cell, T memory Med
FOXP3 Human PCH101 PE-Cy5.5 Treg T cell, Treg Low
IL-22 Human 22URTI PE-Cy7 Effector function T cell, T helper Low
CD3 Human SK7 APC-H7 T cell lineage T cell, T helper, T memory, CTL, Treg, γδ T cell High
CD4 Human SK3 Brilliant Ultraviolet 496 T cell lineage T cell, T helper, T memory, Treg High
CD25 Human 2A3 Brilliant Ultraviolet 563 Treg T cell, Treg Low
CD95 Human Dx2 Brilliant Ultraviolet 737 Differentiation T cell, γδ T cell, T memory, T helper, CTL Low
CD8a Human SK1 Brilliant Ultraviolet 805 T cell lineage T cell, CTL, T memory High
IL-2 Human MQ1-17H12 Brilliant Violet 421 Effector function T cell, CTL, T helper Med
IL-17A Human BL168 Brilliant Violet 605 Effector function T cell, T helper Low
CD69 Human FN50 Brilliant Violet 711 Activation T cell, T memory, T helper, CTL, γδ T cell Med
TNF Human Mab11 Brilliant Violet 750 Activation T cell, T helper, CTL Med
CCR7 Human 150503 Brilliant Ultraviolet 395 Differentiation T cell, T memory, γδ T cell Low
Perforin Human B-D48 Alexa Fluor 594 cytotoxic molecule T cell, CTL, γδ T cell Med
CD39 Human TU66 Brilliant Ultraviolet 661 Differentiation T cell, T helper, Treg Med
Granzyme B Human GB11 FITC Cytotoxic molecule T cell, CTL, γδ T cell High
IFN-gamma Human B27 Brilliant Blue 700 Effector function T cell, T helper, γδ T cell, CTL High
CD107a Human H4A3 Alexa Fluor 700 Degranulation T cell, CTL, γδ T cell Med
CD40L Human TRAP1 Brilliant Violet 480 Co-stimulatory T cell, T helper Med
CD27 Human L128 Brilliant Violet 786 Differentiation T cell, γδ T cell, T memory Med
Ki-67 Human B56 Brilliant Violet 650 Proliferation T cell, T memory, T helper, CTL, γδ T cell Low
IL-21 Human 3A3-N2.1 Alexa Fluor 647 Effector function T cell, CTL, T helper Low
CD45RO Human UCHL1 Brilliant Violet 570 Differentiation T cell, T memory, γδ T cell Med