Anti mouse il 17 fitc

DSS (molecular weight: 6500–10000, No. Lot#SLBB4215V) and Concanavalin A (ConA, No. 71K7024) were purchased from Sigma-Aldrich. Collagenase IV was obtained from Gibco, New York, USA (250 units/mg, No. 1371044). Cell Stimulation Cocktail (containing PMA and ionomycin, No. E13494-112), Intracellular Fixation and Permeabilization Buffer (No. E09680-1642), Anti-Mouse CD4 antigen presenting cell (APC, No. E07036-1634), Anti-Mouse IFN gamma PE (No. E02134-1631), Anti-Mouse IL-17-FITC (No. E00850-1631), Anti-Mouse RORγt mAb (No. E05251-1630), Anti-Mouse Foxp3 mAb, and Anti-Mouse IL-10 PE (No. E02094-1633) were acquired from EBioscience, California, USA. Percoll was purchased from Pharmacia, Uppsala, Sweden, and Dithiothreitol (DTT) was acquired from Promega, Madison, USA. DNase was obtained from Biodee, Beijing, China (No. R0110). PV-9001 Polink-2 plus® Polymer HRP Detection System including 3% H₂O₂, Reagent 1(Polymer Helper) and Reagent 2 (HRP-labeled Goat Anti-Rabbit IgG), HRP-labeled Goat Anti-Mouse IgG, and Mouse anti-β-Actin Monoclonal Antibody were purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, China.

Cell pellets were prepared from spleen tissues. The regulatory T cell populations were examined using anti-mouse CD4–peridin chlorophyll protein (perCP) and anti-mouse CD25-allophycocyanin (APC) (eBioscience); then, the cells were fixed and permeabilized using a Foxp3/Transcription Factor Staining Buffer set (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. For Th17 cell analysis, before FACs staining, the cells were stimulated with 25 ng/mL phosphomolybdic acid (Sigma-Aldrich, St. Louis, MO, USA), 250 ng/mL ionomycin (Sigma-Aldrich), and Golgi Stop (BD Biosciences, San Diego, CA, USA) in 5% CO₂ at 37°C for 4 hours. The cells were stained with anti-mouse CD4 PerCP, and then with an anti-mouse IL-17 FITC (eBioscience), followed by fixation and permeabilization using a Cytofix/Cytoperm Plus Kit (BD Biosciences) according to the manufacturer’s instructions. The samples were analyzed using a FACSCalibur instrument (BD Pharmingen; BD Biosciences).