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OMIP-025

Evaluation of human T- and NK-cell responses including memory and follicular helper phenotype by intracellular cytokine staining

Gemma Moncunill, Carlota Dobano, M. Juliana McElrath, Stephen C. Rosa Cytometry PART A, Volume 87, Issue 4, 289-292 (2015)

PURPOSE: This panel was developed to assess antigen-specific T cells using peptide pools to various antigens of interest, although other types of antigens such as recombinant proteins or whole pathogens could be considered using different stimulation times. In addition to multiple functional markers, the panel includes differentiation markers and markers to assess follicular helper T cells and NK cells (Table 1). It was optimized using cryopreserved peripheral blood mononuclear cells (PBMC) from human immunodeficiency virus (HIV) uninfected and HIV infected adults with known cytomegalovirus (CMV) responses and it underwent assay qualification. The panel is being used to evaluate the responses to HIV and malaria vaccine candidates in adults and children from different geographic areas.

CELL TYPE: Cryopreserved PBMC

MACHINE: BD LSR2

Marker Target Clone Fluorochrome Purpose
Dead cells Human Live/Dead Fix Aqua viability
CD4 Human SK3 BUV395 T cell lineage
IFN-gamma Human B27 V450 Function
CD14 Human M5E2 Brilliant Violet 510 monocyte (dump)
CD3 Human UCHT1 Brilliant Violet 570 T cell lineage
CD40L Human 24-31 Brilliant Violet 605 Function
CD56 Human HCD56 Brilliant Violet 650 NK, NKT-like cells
CD8 Human RPA-T8 Brilliant Violet 711 T cell lineage
ccr7 Human G043H7 Brilliant Violet 785 memory/differentiation
TNF alpha Human Mab11 FITC Function
IL-4 Human MP4-25D2 PerCP-Cy5.5 Function
IL-2 Human MQ1-17H12 PE Function
CD185 Human MU5UBEE PE-eFluor 610 Tfh
PD-1 Human PE-Cy7 Tfh
IL-21 Human 3A3-N2 APC Function
CD45RA Human HI100 APC-H7 memory/differentiation