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30‐Parameter Flow Cytometry Panel to Characterize iNKT, NK, Unconventional and Conventional T Cells

Thomas Liechti, Mario Roederer Cytometry PART A, Volume 95, Issue 9, 946-951 (2019)

PURPOSE: This panel enables the in‐depth immunophenotyping of unconventional CD3+ T‐cell populations such as invariant natural killer T cells (iNKT; CD1d:PBS57 tetramer), γδ T cell subsets (TCR Vδ1, TCR Vδ2, TCR Vγ9), and mucosal‐associated invariant T cells (MAIT; CD161, TCR Vα7.2) in combination with conventional CD3+ T cells (CD4, CD8) and natural killer (NK) cell subsets (CD16, CD56) (Table 1). We included markers to assess differentiation stages (CCR7, CD27, CD28, CD45RA, CD57, and CD95) and expression of cytokine receptors (CD122, CD127), activation markers (CD38, HLA‐DR), chemokine receptors (CCR5, CXCR5), the co‐inhibitory molecule PD‐1, and activating and inhibitory NK receptors (CD158, CD244, and CD318). This unique combination of markers enables a thorough phenotypic characterization of these cell subsets (Table 2).



Marker Target Clone Fluorochrome Purpose
Dead cells Human Live/Dead Fix Blue viability
TCR V delta 1 Human TS8.2 FITC ?d T
CD57 Human NK-1 BB630-P2 Immune senescence
CD244 Human 25235 BB660-P2 Co-stimulatory/inhibitory receptor
CD127 Human hIL-7R-M21 BB700 Cytokine receptor
CD185 Human RF8B2 BB790-P Chemokine receptor
TCR V gamma 9 Human B3 PE ?d T
TCR V delta 2 Human B6 PE-CF594 ?d T
CD161 Human DX12 PE-Cy5 MAIT
HLA-DR Human Tu36 PE-Cy5.5 Activation
CD314 Human 1D11 PE-Cy7 KIR/NKG2D
CD1d Human APC iNKT
CD45RA Human HI100 Alexa Fluor 700 Differentiation
CCR5 Human 2D7/CCR5 APC-Cy7 Chemokine receptor
ccr7 Human 150503 BUV395 Differentiation
CD16 Human 3G8 BUV496 NK
CD56 Human BUV563 NK
PD-1 Human EH12.1 BUV661 Inhitibory receptor
CD95 Human Dx2 BUV737 Differentiation
CD4 Human SK3 BUV805 CD4 T
CD122 Human Mik-b3 Brilliant Violet 421 Cytokine receptor
CD3 Human UCHT1 Brilliant Violet 510 CD3 T
CD8 Human RPA-T8 Brilliant Violet 570 CD8 T
CD158 Human DX27 Brilliant Violet 605 KIR/NKG2D
CD28 Human CD28.2 Brilliant Violet 650 n/a
TCR V alpha 7.2 Human 3C10 Brilliant Violet 711 n/a
CD38 Human HB7 Brilliant Violet 750 n/a
CD27 Human L128 Brilliant Violet 786 n/a