Anti mouse cd25 clone pc61

T cells and their subsets were analyzed by differential expression of surface markers as well as by their intracellular cytokine profile. Briefly, cells were stained with different surface markers, such as anti-mouse CD3e (clone BM10-37, BD Biosciences), anti-mouse CD4 (clone RM4-5, BD Biosciences), anti-mouse CD8a (clone53-6.7, BD Biosciences) and anti-mouse CD25 (clone PC61, BD Biosciences). For analysis of the expression of intra cellular cytokines, cells were stimulated with 50 ng of PMA and 1 μg of ionomycin for 4 h in the presence of Golgi stop (BD Biosciences) to inhibit the secretion of the cytokines from the cells. Cells were then washed with staining buffer, fixed and permealized with BD cytofix and cytoperm^TMPlus (BD Biosciences) along with 0.2% Tween 20 for 20 minutes. Next, cells were stained with anti-mouse IL4 (clone 11B11, BD Biosciences), anti-mouse IFN-γ (clone XMG1.2, BD Biosciences), anti-mouse IL-17A (clone TC11-18H10) and anti-mouse Foxp3 (clone MF23, BD Biosciences). Brilliant stain buffer (BD Biosciences) and ultra comp beads (eBioscience, San Diego) were used for the dilution of the stains and compensation respectively. All the stained cells were analyzed by LSRII flow cytometer (BD Biosciences) and the data was evaluated by FlowJo software.

Cell surface staining was performed according to standard procedures using antibodies against anti-mouse CD4 (clone GK1.5; BD Pharmingen), anti-mouse Thy1.1 (clone OX-7; BD Pharmingen), anti-mouse Thy1.2 (clone 53-2.1; BD Pharmingen), anti-mouse LAG-3 (clone C9B7W; BD Pharmingen), anti-mouse TNFR2 (clone TR75-89; BD Pharmingen), anti-mouse CD25 (clone PC61; BD Pharmingen), anti-mouse CTLA-4 (clone UC10-F10-11; BD Pharmingen), anti-mouse PD-1 (clone J43; eBioscience), anti-mouse GARP (clone YGIC86; eBioscience), anti-mouse CD38 (clone 90; eBioscience), anti-mouse GITR (clone DTA-1; eBioscience), anti-mouse neuropilin-1 (clone 3DS304M; eBioscience), and anti-mouse CD73 (cloneTY/11.8; Biolegend). Foxp3 was detected using the GFP reporter or by intracellular cytokine staining using a anti-mouse Foxp3 (clone FJK-16 s; eBioscience) antibody. All flow cytometry was performed on a BD LSRII or BD FACSCalibur and analyzed using FlowJo (TreeStar). Surface stain was performed by incubating cells with antibody cocktail mix for 20 minutes at 4°C. For intracellular cytokine Foxp3 staining, cells were first stained for surface receptors, fixed in 4% formyl saline, permeabilized (0.5% BSA, 0.1% Triton, and 2 mM EDTA in PBS) for 45 minutes at room temperature. Cells were incubated overnight with the anti-cytokine antibodies and analyzed by flow cytometry.