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NKp46‐Based 27‐Color Phenotyping to Define Natural Killer Cells Isolated From Human Tumor Tissues

Marie Frutoso, Florian Mair, Martin Prlic Cytometry PART A, Volume 97, Issue 10, 1052-1056 (2020)

PURPOSE: This 27‐color panel has been validated and optimized to comprehensively profile natural killer (NK) cells isolated from human tumors using a collagenase Type II‐based digestion protocol. We confirmed that detection of protein expression by antibodies used in our final panel was not affected during tissue digestion. During this evaluation process, we found that detection of CD56, a biomarker typically used to identify NK cells, was affected substantially by collagenase‐based digestion. Thus, our panel is centered around expression of NKp46, which is sufficient to identify NK cells and not affected by the tissue collagenase digestion step. Our panel further includes biomarkers used to extrapolate NK‐cell maturation, differentiation, migration, homing potential, and functional state. Our panel is intended to provide in‐depth characterization of human NK cells isolated from tissues, which we specifically tested using oral squamous cell carcinomas tissues, but it is compatible with other tissues that can be dissociated with a collagenase Type II‐based protocol.

CELL TYPE: Tumor tissues and peripheral blood. Oral squamous cell carcinoma tissues with donor‐matched blood were used to profile NK cells with this 27‐color panel


Marker Target Clone Fluorochrome Purpose
Dead cells Human Live/Dead Fix Blue viability
CD45 Human HI30 BUV805 lymphocyte
CD14 Human M5E2 Brilliant Violet 570 monocyte
CD19 Human BUV395 B
CD3 Human UCHT1 BUV661 T
CD127 Human hIL-7R-M21 Brilliant Violet 786 ILC
HLA-DR Human APC-H7 exclusion/activtion
CD16 Human 3G8 BUV496 NK and ADCC
NKp46 Human 900 Biotin NK and activation
Streptavidin Human BB630-P2 secondary reagent to NKp46
CD314 Human 1D11 PE-Cy7 activation
CD244 Human 25235 Brilliant Violet 421 activation
CD2 Human RPA-2.10 Brilliant Violet 605 co-stimulatory
CD159a (NKG2a) Human REA110 PE inhibitory
CD161 Human DX12 BB700 inhibitory
TIGIT Human 741182 Brilliant Violet 711 co-inhibitory
CD69 Human FN50 Brilliant Violet 650 residency and activation
CD39 Human TU66 PE-CF594 residency and activation
CD57 Human NK-1 FITC maturation and differentiation
CD27 Human M-T271 BB660-P2 maturation
CD11b Human ICRF44 PE-Cy5 maturation and adhesion
CD38 Human HIT2 BB790-P activation
CX3CR1 Human 2A9-1 BUV737 migration
CD103 Human Ber-ACT8 Brilliant Violet 750 residency
Ki-67 Human SolA15 eFluor 660 proliferation
Granzyme B Human Alexa Fluor 700 cytolytic ability
CD25 Human 2A3 BUV563 activation
CD56 Human CMSSB PE-Cy5.5 activation, NK for PB