Bm cytofix buffer and perm wash reagent

Peripheral lymph nodes (LNs) and spleens from wild type mice were harvested and mashed through a 70 micron strainer. CD4+ T cells were enriched by positive selection using CD4+ MACS beads (Miltenyi). T cells (5 × 10⁵) were then cultured for 4 days in 24-well plates with plate-bound anti-CD3 (2 ug/mL), soluble anti-CD28 (1 ug/mL), and recombinant TGF-β (2 ng/mL) and the indicated BMDM supernatants. On day 4, T cells were stimulated with PMA/iononmycin and brefeldin A (BD Biosciences) for 4 hr. Cells were stained with fixable viability dye (eFluor780; eBiosciences) to exclude dead cells, surface stained with Abs to CD4 (RM4-5) and TCRβ (H57-597), treated with BM Cytofix Buffer and Perm/Wash reagent (BD Biosciences), and stained with anti-IL-17A (17B7). Antibodies were purchased from BD Biosciences or eBiosciences.

Cells were stained with Abs to CD4 (RM4-5), TCRβ (H57-597), IL-17A (17B7), CD11b (M1/70), or Ly6G (1A8). For mitochondrial studies, cells were treated as indicated and then stained with 30 minutes with Mitotracker Deep Red (100nM), Mitotracker Green (100nM), TMRM (100nM) or MitoSOX (2.5 uM) at 37°C. All mitochondrial staining reagents were from Life Technologies. For intracellular cytochrome C staining, cells were stimulated as indicated and then treated on ice with either control or digitonin (25 ug/ml) for 10 minutes. Cells were then treated with BM Cytofix Buffer and Perm/Wash reagent (BD Biosciences), and stained with anti-cytochrome C (6H2.B4). Antibodies were purchased from BD Biosciences or eBiosciences.