Fig. 3
From: Simplified assay for enrichment of primed human Th17 and Tc17 lymphocytes from peripheral blood
IL-17A secreting cells are enriched within CD146+ CD4 and CD146+ CD8 subsets. Lymphocytes from whole blood lysis of healthy human peripheral blood were stained with mouse anti Human mAb CD4, CD8, CD45 RO and CD146 as described and sorted using flow cytometer. Sorted cells were finally washed with IMDM media and plated in 96 well flat bottom culture plate coated with purified anti-human CD3 and CD28 antibodies. Cells were incubated for 5 days; cultured cells were re-supplemented for the last 4 h of incubation with IMDM + 10% FCS and PMA/Ionomycin in the presence of brefeldin-A and collected to perform intracellular cytokine staining for IL-17 and IFN-γ cytokines