MOESM1 of New insights into the heterogeneity of Th17 subsets contributing to HIV-1 persistence during antiretroviral therapy
2016-08-24T05:00:00Z (GMT) by
Additional file 1. Purity of flow cytometry-sorted memory CD4+ T-cell subsets. Total CD4+ T-cells were isolated from PBMCs of healthy individuals by negative selection using magnetic beads (Miltenyi). Cells were stained with a cocktail of fluorochrome-conjugated Abs and analyzed by polychromatic flow cytometry (see Supplemental Experimental Procedure). Memory (CD45RA−) cells lacking the lineage-specific markers CD8 (CD8+ T-cells), CD19 (B cells), and CD56 (NK cells) and with differential expression of CCR6, CCR4, and CXCR3 were sorted by flow cytometry (BDAria II) as follows: CCR6+CCR4+CXCR3− (Th17), CCR6+CCR4−CXCR3− (CCR6+DN), CCR6+CCR4+CXCR3+ (CCR6+DP), CCR6+CCR4−CXCR3+ (Th1Th17) and CCR6−CCR4−CXCR3+ (Th1). A viability staining was used to exclude dead cells. The positivity gates where defined based on fluorescence minus one (FMO) controls. Shown is (A) the gating strategy for the identification of different subsets on CD4+ T-cells sorted by MACS and (B) purity upon FACS sorting of different memory T-cell subsets. The percentage of each subset is indicated on the figures. Results are from one donor representative of experiments performed with cells from >10 different donors. The positivity gates where defined based on fluorescence minus one (FMO) controls.