Phosphorylation Analysis of Tregs

Flow cytometry was performed with directly conjugated Abs as previously described (6 (link)). A fixable Blue LIVE/DEAD dye (Life Technologies) was used to exclude dead cells from analysis. FOXP3 and CTLA-4 (total) staining was performed using the eBioscience FOXP3 staining buffers. For staining of phosphorylated proteins, BD Fix Buffer I and BD Phosflow perm buffer III were used according to manufacturer’s instructions. In brief, sorted Tregs (at 5 × 10⁵/ml in 100 μl RPMI 1640) were incubated for 15 min with either SF or 10 ng/ml rhIL-6 before addition of 100 μl BD Fix Buffer I and incubated for 10 min at 37°C. Cells were washed extensively in PBS, then permeabilized by addition of 1 ml ice-cold Perm Buffer III and incubation for 30 min on ice. Cells were washed three times and then stained for PE–anti–p-STAT3 or PE–anti–p-STAT5. Data were collected on the LSR II, FACS Aria, or FACSCalibur flow cytometers (all BD Biosciences). Flow cytometry data were analyzed using FloJo (Tree Star) software.