Cd45 apc hi30

Cell suspensions were stained with optimal quantity of antibodies at a concentration of 10⁷ cells/ml in a final volume of 100μl of PBS/FCS 3%. Incubation was performed in the dark at 6°C for 20 min. The following mAbs were used for cell surface staining: anti-CCR5-APC (2D7; BD Pharmingen), anti-CD4 PerCP (RPA-T4, Biolegend), anti-CD8 A700 or Pacific Blue (HIT8a, RPA-T8, Biolegend), anti-CD8 PE (DK25, DAKO), CD45 APC (HI30, Biolegend), CD3 PeCy7 (UCHT1, Biolegend), CD45 PE-CF594 (HI30, BD Pharmingen). All cell preparations were acquired on an LSRII cytometer (BD) and analyzed with FlowJo software (Tree Star, Portland, OR). Frequencies of positive cells were determined according to the Fluorescence minus one (FMO) staining control. Absolute counts in blood were determined by cell-surface staining in whole blood using TrueCount beads, as recommended by the manufacturer (BD).

PBMC and BALF samples were incubated with Fc-receptor blocking reagent (BD Biosciences) at room temperature for 15 min and surface stained with the following fluorochrome-conjugated antibodies at 4°C for 40 min: CD45-APC (HI30, BioLegend), CD3-APC-cy7 (UCHT1, BioLegend), CD8-BV510 (SK1, BioLegend), CD4-BV421 (RPA-T4, BD Biosciences), CD161-PE (HP-3G10, BioLegend), CD161-BV421 (HP-3G10, BioLegend), Vα7.2-PE-cy7 (3C10, BioLegend), CD69-FITC (FN50, BioLegend), CD69-PE (FN50, BioLegend), and PD-1-BV421 (EH12.1, BD Biosciences). MAIT cells were identified as CD3⁺CD161^high Vα7.2⁺ cells.
For intracellular cytokine staining, PBMCs were cultured in RPMI 1640 supplemented with 10% FBS in the presence of phorbol 12-myristate 13-acetate (PMA) (25 ng/ml) and ionomycin (500 ng/ml) for 40 min followed by a 3.5-h incubation with brefeldin A in a 5% CO₂ incubator at 37°C. IL-17A- and/or IFN-γ-producing cells were identified by intracellular staining with anti-IL-17A-FITC (BL168, BioLegend) and anti-IFN-γ-FITC (4S.B3, BioLegend) for 45 min after surface staining and fixation/permeabilization. Fluorescence Minus One (FMO) was used as negative control; the gating strategy for surface markers and intracellular cytokines is shown in Supplemental Figure 1. Cells were acquired by flow cytometry using a FACSVerse (BD Biosciences) and analyzed with FlowJo software (TreeStar).