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A 31-Parameter Flow Cytometry Panel for In-Depth Immunophenotyping of Human T-Cell Subsets Using Surface Markers

Song-Rong Wang, Na Zhong, Xin-Mei Zhang, Zhi-Bin Zhao, Robert Balderas, Liang Li, Zhe-Xiong Lian Cytometry PART A, Volume 99, Issue 3, 273-277 (2021)

PURPOSE: Dissecting the functional diversity of T cells is critical in elucidating mechanisms and in developing therapies for various diseases. Here, we designed a 31-parameter (29-color) panel to enable the characterization of T-cell subsets and immunophenotyping of the human peripheral blood and lymph nodes using cell surface staining. In addition to adaptive T-cell markers, TCR Vα24-Jα18, TCR γδ, TCR Vɑ7.2, and CD161 were included to identify iNKT, γδ T, and MAIT cells, respectively, which are innate-like T cells. C-X-C chemokine receptors (CXCR3, CXCR4, CXCR5, CXCR6) and C-C motif chemokine receptors (CCR4, CCR6, CCR7) were included to enable the identification of Th cell subsets (Th1, Th2, Th17), Tfh cell subsets (Tfh1, Tfh2, Tfh17), and Th cells with specific homing capacities. Furthermore, in this panel, we also used markers for assessing cell differentiation (CD45RO, CD7), activation (CD57, CD95, HLA-DR) and the expression of some cosignaling molecules (PD-1, NKG2D, CD28). Particularly, CD69 and CD103 were included for the further analysis of tissue resident memory T (Trm) cells. This panel would enable the in-depth immunophenotyping of human T-cell subsets, and may be applied in the monitoring, prognosis, and mechanistic studies of various immune-related diseases.



Marker Target Clone Fluorochrome Purpose
Dead Cell All Species Zombie UV viability
CD3 Human UCHT-1 BUV805
CD4 Human SK3 Brilliant Violet 750
CD7 Human M-T701 PE-Cy7
CD8 Human RPA-T8 Brilliant Violet 786
CD25 Human 2A3 Brilliant Violet 421
CD28 Human CD28.2 PE
CD45 Human HI30 APC-R700
CD45RO Human UCHL1 PE-CF594
CD56 Human NCAM16.2 Brilliant Violet 650
CD161 Human DX12 BUV737
PD-1 Human EH12.1 BB515
HLA-DR Human G46-6 Brilliant Violet 605
TCR delta/gamma Human APC
CD183 Human G025H7 Brilliant Violet 510
CXCR4 Human 12G5 BUV563
CD186 Human 13B 1E5 BB700
CCR6 Human 11A9 BUV496
TCR V alpha 24 J alpha 18 Human 6B11 Brilliant Violet 711
TCR V alpha 7.2 Human OF-5A12 Brilliant Violet 570
CD57 Human NK-1 Brilliant Violet 480
CD103 Human 2E7 BB630-P2
CD314 Human 1D11 BB660-P2
CD185 Human RF8B2 BUV615
CD194 Human [1G1] BUV395
CD95 Human DX2 BUV395
CCR7 Human 2-L1-A APC-Cy7
CD69 Human FN50 BB790-P
CD127 Human HIL-7R-M21 PE-Cy5