Cell cycle analysis
Sorted Th17 subpopulations were fixed in ice-cold 70% ethanol for 2.5 hours. Cells were centrifuged for 5 min at 300 rcf at 4oC. Ethanol was removed and cells were rinsed once with ice-cold Hank’s balanced salt solution (HBSS) containing Mg2+ and Ca2+. After that, cells were suspended in 0.5 ml of ice-cold HBSS containing Mg2+ and Ca2+. The cell suspension was mixed with an equal volume of HBSS containing 2 mg Hoechst 33342 (Molecular Probes) and 4 mg Pyronin Y (Sigma-Aldrich) and incubated for 20 minutes in the dark. Cells were then loaded on the flow cytometer for cell cycle analysis.