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
Cytometry PART A, Volume 97, Issue 10, 1019-1023 (2020)
Nina Hertoghs, Katharine V. Schwedhelm, Kenneth D. Stuart, Margaret Juliana McElrath, Stephen C. De Rosa
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 | Fluorophore | Purpose |
---|---|---|---|---|
CD16 | Human | 3G8 | Brilliant Ultraviolet 395 | NK, monocyte, ILC |
Dead cells | Human | Live/Dead Fix Blue | Viability | |
CD3 | Human | UCHT1 | Brilliant Ultraviolet 496 | T cell |
CD19 | Human | SJ25C1 | Brilliant Ultraviolet 563 | B cell |
HLA-DR | Human | G46-6 | Brilliant Ultraviolet 661 | Activation |
CD27 | Human | L128 | Brilliant Ultraviolet 737 | NK phenotyping |
CD8 | Human | SK1 | Brilliant Ultraviolet 805 | 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 | Brilliant Blue 660-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 | Brilliant Blue 790-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 |