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28‐Color Flow Cytometry Panel for Broad Human Immunophenotyping

Kathryn Payne, Wenyan Li, Robert Salomon, Cindy S. Ma Cytometry PART A, Volume 97, Issue 8, 777-781 (2020)

PURPOSE: A 28-color panel was developed to screen for a range of lymphocyte subsets in human peripheral blood mononuclear cells (PBMCs), particularly in patients with primary immunodeficiency (PID). Using the panel, we are able to avoid running the sample over multiple screening panels while still deeply phenotyping a diverse range of lymphocyte subsets including innate like lymphocytes (γδ, mucosal-associated invariant T [MAIT], natural killer [NK], and NKT cells), as well as multiple subsets of naïve and memory CD4+ and CD8+ T cells, and B cells. Specifically, naïve, central memory (cmem) and effector memory (emem) CD4+ T cells, naïve cmem, emem, and CD45RA+ revertant memory (TEMRA) CD8+ T cells, regulatory (Tregs), T follicular helper (Tfh) and T helper (Th) 1, and Th17 CD4+ T cells, and transitional, naïve, memory, and CD21 B cells.



Marker Target Clone Fluorochrome Purpose
Dead cells Human Zombie UV Viability
CD20 Human 2H7 BUV805 B Cell
TCR alpha/beta Human T10B9.1A-31 BUV737 T cell (conventional aß T)
CD4 Human SK3 BUV661 T helper
CD185 Human RF8B2 BUV615 Tfh
CD21 Human B-ly4 BUV563 B Cell (Down-regulated on atypical/aged memory B cells)
CD8 Human RPA-T8 BUV496 Cytotoxic T cell
CD45RA Human HI100 BUV395 naïve and revertant effector (TEMRA) T cells
CD161 Human DX12 Brilliant Violet 786 MAIT, NK, NKT, and a subset of CD8+ T cells
CD19 Human SJ25C1 Brilliant Violet 750 B Cell
TCR gamma/delta Human 11F2 Brilliant Violet 711 ?d T cells
CD10 Human HI10a Brilliant Violet 650 Transitional B cells
CD3 Human UCHT1 Brilliant Violet 570 T cell
IgD Human IA6-2 Brilliant Violet 480 Naive B cells, low levels on IgM memory B cells
CD183 Human 1C6 Brilliant Violet 421 Th1
CD56 Human NCAM16.2 BB790-P NK cells and some memory CD8+ T cells
CD127 Human hIL-7R-M21 BB700 Down-regulated on Tregs
IgG Human G18-145 BB660-P2 IgG+ Isotype switched memory B cells
CCR6 Human 11A9 BB630-P2 Th17
CD27 Human M-T271 BB515 Memory B cells; naïve and cmem CD4+ and CD8+ T cells
ccr7 Human G043H7 PE-Cy7 Naïve T and CMEM T cells
IgA1/IgA2 Human G20-359 PE-Cy5 IgA+ Isotype switched memory B cells
PD-1 Human EH12.1 PE-CF594 Activation
CD25 Human 2A3 BYG584-P Treg
TCR V alpha 7.2 Human 3C10 APC-Cy7 T cell receptor for MAIT cells
IgM Human G20-127 APC-R700 Naïve B cells and IgM+ memory B cells
KLRG1 Human 2F1/KLRG1 APC senescence on CD8+ T cells