Anti mouse foxp3 phycoerythrin pe

Before cell staining, differentiated CD4⁺ T cells were stimulated with 25 ng/ml phorbol myristate acetate and 250 ng/ml ionomycin (both from Sigma, St. Louis, MO, USA) in the presence of GolgiStop (BD Pharmingen, San Diego, CA, USA) for 4 hours. The cells were stained with anti-mouse CD4 peridin chlorophyll protein (PerCP), anti-mouse CD25 allophycocyanin (APC), anti-mouse IL-17 fluorescein isothiocyanate (FITC), and anti-mouse FOXP3 phycoerythrin (PE) (eBiosciences, San Diego, CA, USA) followed by fixation and permeabilization with a Foxp3 staining buffer kit according to the manufacturer’s instructions to examine intracellular cytokines. All samples operated on a FACSCalibur (BD Pharmingen) and data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).

Stimulated T cells and IL-12 receptor-specific siRNA-transfected T cells were stained with anti-mouse CD4-peridinin-chlorophyll protein and anti-mouse CD25-allophycocyanin for 30 min at 4 °C. The cells were permeabilized and fixed with CytoPerm/CytoFix (BD Biosciences, San Diego, CA, USA) in accordance with the manufacturer’s protocol; further stained with anti-mouse Foxp3-phycoerythrin (PE) and/or anti-mouse IL-17-fluorescein isothiocyanate (FITC), anti-mouse IFN-γ-PE, and anti-mouse IL-4-PE (all from eBioscience, San Diego, CA, USA); and subjected to flow cytometric analysis (FACSCalibur; BD Biosciences). For p-STAT3 Y705 and S727 and p-STAT5 analysis, splenocytes were treated with p40-EBI3 (1 and 10 μg/mL) and IL-6 for 1 h; the cells were then stained with anti-mouse CD4-FITC. The cells were fixed and permeabilized with Lyse/Fix Buffer (BD Pharmingen, San Jose, CA, USA) and Perm Buffer II (BD Pharmingen). The permeabilized cells were stained with anti-p-STAT3 Y705-PE, anti-p-STAT3-S727-PE, or anti-p-STAT5-PE (BD Pharmingen) and subjected to flow cytometric analysis (CytoFLEX; Beckman Coulter, Fullerton, CA, USA).