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OMIP-064

A 27‐Color Flow Cytometry Panel to Detect and Characterize Human NK Cells and Other Innate Lymphoid Cell Subsets, MAIT Cells, and γδ T Cells

Nina Hertoghs, Katharine V. Schwedhelm, Kenneth D. Stuart, Margaret Juliana McElrath, Stephen C. De Rosa Cytometry PART A, Volume 97, Issue 10, 1019-1023 (2020)

PURPOSE: This 27‐color flow cytometry panel was developed in order to assess immunological changes over the course of an immunization and challenge regimen in two experimental malaria vaccine trials. The aim of the study was to find correlates of vaccine‐induced protection. Several studies have indicated that protection against malaria appears to involve immune responses at various immunological sites, with liver‐resident responses playing an essential role. As it is not feasible to monitor the immune responses within the liver in humans, this panel is developed with the aim to thoroughly characterize the immune responses over time in blood in addition to detecting changes that might reflect what happens in other immunological sites like the liver. The focus of this panel is to detect several innate lymphoid cell populations, including NK cells and their activation status. Moreover, unconventional T cells like mucosal associated invariant T cells and γδ T cells are assessed in the panel.

CELL TYPE: Cryopreserved peripheral blood mononuclear cells

MACHINE: BD FACSymphony A5

Marker Target Clone Fluorochrome Purpose
CD16 Human 3G8 BUV395 NK, monocyte, ILC
Dead cells Human Live/Dead Fix Blue Viability
CD3 Human UCHT1 BUV496 T cell
CD19 Human SJ25C1 BUV563 B cell
HLA-DR Human G46-6 BUV661 Activation
CD27 Human L128 BUV737 NK phenotyping
CD8 Human SK1 BUV805 CD8 T CELL
TCR gamma/delta Human 11F2 Brilliant Violet 421 Pan-?d T
CD314 Human 1D11 Brilliant Violet 480 NK phenotyping
CD56 Human HCD56 Brilliant Violet 570 NK phenotyping
TCR V delta 2 Human B6 Brilliant Violet 605 ?d T cell subset vd2
CD38 Human HB-7 Brilliant Violet 650 Activation
Ki-67 Human B56 BB660-P2 Proliferation
CD4 Human SK3 Brilliant Violet 750 CD4 T
TCR V alpha 7.2 Human 3C10 Brilliant Violet 786 MAIT
Fc epsilon R1 alpha Human polyclonal FITC NK phenotyping
CD14 Human MfP9 PerCP-Cy5.5 Monocyte
CD57 Human HNK-1 Brilliant Violet 711 NK phenotyping
CD127 Human hIL-7R-M21 BB790-P ILC phenotyping
NKp46 Human 9E2/NKp46 PE NK phenotyping
CD294 Human BM16 PE-CF594 ILC phenotyping
CD33 Human WM53 PE-Cy5 Granulocyte/monocyte exclusion
CD117 Human 104D2 PE-Cy5.5 ILC phenotyping
CD159a (NKG2a) Human Z199 PE-Cy7 NK phenotyping
CD337 Human p30-15 Alexa Fluor 647 NK phenotyping
CD159c Human 134591 Alexa Fluor 700 NK phenotyping
CD161 Human HP-3G10 APC-Fire 750 MAIT/ILC phenotyping