Accurri c6 cytometer

Flow cytometric analysis was performed as described recently [31 (link)]. In brief, single-cell suspensions were labeled directly with the following fluorochrome-conjugated antibodies purchased from either BioLegend, BD Biosciences or eBioscience: anti-human CD2 (RPA-2.10 and TS118), anti-mouse CD3 (17A2), anti-mouse CD4 (RM4-5), anti-mouse CD8α (53-6.7), anti-mouse CD11c (N418), anti-mouse CD19 (6D5), anti-mouse CD44 (IM7), anti-mouse CD45 (30-F11), anti-mouse CD45R (RA3-6B2), anti-mouse CD62L (MEL-14), anti-mouse CD127 (A7R34), anti-mouse Gata3 (L50-823 and TWAJ), anti-mouse Gr1 (RB6-8C5), anti-mouse Ly6G (1A8), anti-mouse MHCII (M5/114.15.2), anti-mouse RORγt (Q31-378), anti-mouse T-bet (4B10) and anti-mouse Ter119 (TER-119). Live/Dead discrimination was carried out using LIVE/DEAD Fixable Blue Dead Cell Stain (Thermo Fisher Scientific). Surface staining was performed for 15 min on ice in PBS (Gibco) containing 0.2% BSA. Intracellular stainings for T-bet, Gata3 and RORγt were performed with the Foxp3/Transcription Factor Staining Buffer Set (eBioscience) according to the manufacturer’s instructions. Flow cytometry samples were acquired at the LSR Fortessa flow cytometer with Diva software (BD Biosciences) and data were analyzed using FlowJo software (Tree Star). Absolute cell numbers were counted with the Accurri C6 Cytometer (Beckton Dickinson) (Supplementary Table 1).

Fluorochrome-conjugated anti-human CD2 (RPA-2.10), anti-CD4 (RM4-5), anti-CD8α (53-6.7), anti-CD11c (N418), anti-CD45R (RA3-6B2), anti-CD62L (MEL-14), anti-F4/80 (BM8), anti-Foxp3 (FJK-16S), anti-IFNγ (XMG1.2), anti-IL-10 (JES5-16E3), and anti-IL-17A (eBio17B7) antibodies were purchased from BioLegend and eBioscience. Prior to cytokine staining, cells were stimulated with phorbol 12-myristate 13-acetate (PMA, 10 ng/mL) and Ionomycin (0.5 μg/mL) for 4 h, with Brefeldin A (10 μg/mL) added for the final two h (all Sigma-Aldrich). Surface staining was performed for 15 min on ice in PBS (Gibco) containing 0.2 % bovine serum albumin (BSA, Sigma-Aldrich). Intracellular Foxp3 staining was performed using Foxp3 staining buffer set (eBioscience) according to the manufacturer's instructions. Absolute cell numbers were determined using Accurri C6 Cytometer (Becton Dickinson). Dead cell exclusion was carried out using LIVE/DEAD Fixable Blue Dead Cell Stain (Thermo Fisher Scientific). Cells were washed, resuspended in phosphate buffered saline/bovine serum albumin (PBS/BSA), and measured at LSR-II SORP with Diva software 6.1 (BD Biosciences). Data were analyzed using FlowJo software (Tree Star).