Cytokine Expression Analysis by Flow Cytometry

IL-17a and IFN-γ cytokine expression were assessed by flow cytometry as described previously^{45 (link)}. Briefly, the cells were stimulated for 4 h in phorbol 1,2-myristate 1,3-acetate (5 ng/ml) and ionomycin (1 µg/ml) in the presence of protein transport inhibitor GolgiPlug (BD Pharmingen). Cells were stained for surface markers with following antibodies: anti-mouse TCRβ (H57–597; eBioscience), anti-mouse CD4 (RM4–5; eBioscience), anti-mouse CD45 (30-F11, eBioscience). Cells were washed and fixed using Cytofix/Cytoperm buffer (BD Pharmingen or eBioscience). Intracellular staining was performed with following antibodies: anti-mouse IL-17a (TC11-18H10.1; BioLegend), anti-mouse IFN-γ (XMG1.2, eBiosciences), anti-mouse Foxp3 (FJK-16s, eBioscience) and anti-human/mouse T-bet (eBio4B10, eBiosciences). Dead cells were excluded from analysis using Zombie Yellow Fixable Viability Kit (Biolegend) or LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Life Technologies).