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28‐Color Flow Cytometry Panel to Evaluate Human T‐Cell Phenotype and Function

Phillip A. Swanson II, Robert A. Seder Cytometry PART A, Volume 97, Issue 10, 1032-1036 (2020)

PURPOSE: This 28-color flow cytometry panel was developed to detect frequencies and function of a broad spectrum of T-cell subsets from the blood of infants and adults following antigen-specific stimulation. This panel includes markers to characterize αβ and γδ T-cell subsets, T regulatory cells (Tregs), T follicular helper cells (TfH), and recent thymic emigrants (RTEs). Additionally, antibodies that measure cell surface activation molecules (CD154, CD69, HLA-DR, and CD38) and function through detection of cytokines (IL-8, IL-2, IL-4, IL-5, IL-13, IL-21, IFNγ, and TNF) are also included. This panel was optimized using PMA-stimulated cryopreserved healthy adult and infant PBMCs as well as stimulation with CMV-specific peptides. Currently, this panel is being used to evaluate antigen-specific T-cell responses in adults and infants following immunization with an attenuated malaria vaccine (Table 1).

CELL TYPE: Cryopreserved PBMC


Marker Target Clone Fluorochrome Purpose
Dead cells Human Live/Dead Fix Blue Viability
ccr7 Human G043H7 Alexa Fluor 700 Differentiation
CD3 Human UCHT1 BUV496 Lineage
CD4 Human SK3 BUV805 Lineage
CD8 Human RPA-T8 PE Lineage
CD25 Human BC96 BB700 Differentiation
CD27 Human O323 Brilliant Violet 785 Differentiation
CD31 Human WM59 Brilliant Violet 711 Differentiation
CD38 Human HIT2 BUV661 Activation
CD45RA Human Brilliant Violet 570 Differentiation
CD69 Human FN50 BB790-P Activation
CD127 Human A019D5 PE-Cy7 Differentiation
CD40L Human 24-31 Brilliant Violet 605 Co-stimulatory
CX3CR1 Human 2A9-1 Brilliant Violet 650 Differentiation
CD183 Human G025H7 PE-Dazzle 594 Trafficking
CD185 Human RF8B2 BUV563 Trafficking
HLA-DR Human Tu36 PE-Cy5.5 Activation
IFN-gamma Human B27 BUV395 Function
IL-2 Human MQ1-17H12 BUV737 Function
IL-4 Human MP4-25D2 Brilliant Violet 421 Function
IL-5 Human TRFK5 Brilliant Violet 421 Function
IL-8 Human G265-8 BB630-P2 Function
IL-13 Human JES10-5A2 Brilliant Violet 421 Function
IL-21 Human 3A3-N2.1 Alexa Fluor 647 Function
PD-1 Human EH12.2H7 BB660-P2 CO-inhibitory
TCR gamma/delta Human REA591 APC-Vio770 Lineage
TCR V gamma 9 Human PE-Cy5 Lineage
TCR V delta 1 Human TS8.2 FITC Lineage
TCR V delta 2 Human 123R3 VioGreen Lineage
TNF alpha Human Mab11 Brilliant Violet 750 Function