Fitc conjugated anti cd95

Mononuclear cells were isolated from the spleen and intestinal lamina propria as previously described (Reiley et al., 2006 (link); Chang et al., 2012 (link)). T_reg cells were purified by flow cytometric cell sorting based on their expression of CD25 and the YFP marker from WT-R26^YFP or Traf3^Treg-KO-R26^YFP mice. Flow cytometric analyses and cell sorting were performed (Reiley et al., 2006 (link)) using a FACSCalibur (BD) and FACSAria (BD), respectively. For intracellular cytokine staining (ICS), cells were stimulated with 50 ng/ml PMA plus 750 ng/ml ionomycin for 4–6 h in the presence of 10 µg/ml monensin, and the fixed cells were incubated with the indicated antibodies and subjected to flow cytometry. FITC-conjugated anti-CD95 (BD); PE-conjugated anti–PD-1, anti-CD138, anti–Bcl-6, anti-CTLA4, and anti-CXCR3 (BD); allophycocyanin (APC)-conjugated anti-GL7 and anti-CXCR5 (BD Bioscience); and PE-Cy7–conjugated anti-CD8 and anti-CD4 (eBioscience) were used. Other antibodies were described previously (Chang et al., 2012 (link)).