Flow cytometry

Cells were stimulated for 4 h with phorbol myristate acetate (PMA; 50 ng/ml; Sigma-Aldrich), ionomycin (1 µg/ml; Sigma-Aldrich), and the Golgi-traffic inhibitor Brefeldin (1 µl/ml: BD Biosciences, Franklin Lakes, NJ, USA). Next, cells were stained with anti-CD3-FITC (145-2C11) (BioLegend), anti-CD4-PerCP/Cy5.5 (RM4-4) (BioLegend), fixable viability dye eFluor 780 (eBioscience), fixed and permeabilized using Cytofix/Cytoperm (BD Biosciences), followed by intra-cellular staining with anti-IL-17-PE (TC11-18H10.1) (BioLegend), anti-FOXP3-Alexa Fluor 647 (150D) (BioLegend) and anti-RORγt-BV421 (Q31-378) (BD Biosciences). The gating of T cells was as follows. First, cells were gated in an FSC-A vs. SSC-A dot plot, and subsequently gated in FSC-W vs. FSC-A and SSC-W vs. SSC-A dot plots to eliminate doublets. Living cells were gated based on FVD, and next CD3 + /CD4 + cells were gated on the basis of FMO controls. Finally, IL-17 + and FOXP3 + cells were determined in this gate as markers for Th17 and Treg cells, respectively. Cells were analyzed on the Cytoflex and subsequently using Kaluza software version 2.1.