Anti mouse ifn gamma pe

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.

Lymphocytes were separated from spleen tissue samples using lymphocyte separation medium (Solarbio Biotec, Beijing). The cells were counted, and 5 × 10⁵ to 1 × 10⁶ cells were harvested in cold PBS. The cells were then resuspended in 100 μl PBS and incubated with 0.5 μg anti-mouse CD3a PE, 0.25 μg anti-mouse CD4 FITC, 0.125 μg anti-mouse IFN gamma PE, 0.25 μg anti-mouse IL-4 FITC, 1 μg anti-mouse/rat Foxp3 FITC, 0.25 μg anti-mouse IL-10 FITC or isotype control antibodies (eBioscience, San Diego, CA). The cells were then washed with cold PBS, and 10⁴ cells per sample were analyzed by flow cytometry (CyFlow, PARTEC).