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16-color panel to measure inhibitory receptor signatures from multiple human immune cell subsets

Anna C. Belkina, Jennifer E. Snyder-Cappione Cytometry PART A, Volume 91, 175–179 (2016)

PURPOSE: The panel was developed to determine the combinational inhibitory receptor expression (“IR signatures”) of CD4+ T cells, CD8+ T cells, Natural Killer (NK) cells, invariant Natural Killer T (iNKT) cells, and gamma delta (γδ) T cells from individual human samples. The inhibitory receptors measured are PD-1, TIM-3, CD160, LAG-3, and TIGIT. The activation marker CD137 (4-1BB) is also included in the panel, as is IL-7Rα (CD127). This panel works well with cryopreserved PBMC from healthy and HIV-infected individuals well as fresh tumor specimens. For optimum performance of this panel with digested tumor specimens, the pan-lymphocyte marker CD45 is included (swapped for LAG-3). Other tissues/sample types have not yet been evaluated. Also, a modification of this panel has been optimized that includes CD45RO and CD25 in place of CD160 and CD137.

CELL TYPE: Immune Cells


Marker Target Clone Fluorochrome Purpose
Dead cells Human Zombie NIR Viability
TCR gamma/delta Human B1 BUV395 ?d T cell
CD127 Human hIL-7R-M21 BUV737 Exhaustion, Treg
CD8 Human SK1 BUV805 Lineage
PD-1 Human EH12.2H7 Brilliant Violet 421 Exhaustion
CD3 Human OKT3 Brilliant Violet 510 T cell lineage
CD16 Human 3G8 Brilliant Violet 605 NK lineage/subsets
CD137 Human 4B4-1 Brilliant Violet 650 Acivation
CD56 Human NCAM16.2 Brilliant Violet 786 NK lineage/subsets
CD160 Human BY55 Alexa Fluor 488 Exhaustion
TCR V alpha 24 Human C15 PE iNKT lineage
Lag-3 Human 3DS223H PE-eFluor 610 Exhaustion
TIGIT Human MBSA43 PerCP-eFluor 710 Exhaustion
TIM-3 Human F38-2E2 PE-Cy7 Exhaustion
CD1d Human APC iNKT lineage
CD4 Human RPA-T4 Alexa Fluor 700 Th lineage
CD14 Human hCD14 APC-Cy7 monocyte exclusion
CD19 Human HIB19 APC-Cy7 B cell exclusion
CD45 Human HI30 PE-Dazzle 594 hematopoitic cell ineage
CD25 Human BC96 Brilliant Violet 605 Treg, T cell activation
CD45RO Human UCHL1 Brilliant Violet 650 naïve/memory
CD16 Human 3G8 FITC NK cell lineage, subsets