Anti mouse foxp3 mab

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.

Splenocytes were incubated with 2.4G to block FcRs and then incubated with optimal concentration of fluorochrome-labeled mAbs for 30 minutes at 4°C in dark [10 (link)]. Cells were washed three times and resuspended by FCM buffer (PBS with 0.1% BSA and 0.1%NaN₃). At least ten thousand cells were assayed using a FASCalibur flow cytometry (Becton Dickinson, CA), and data were analyzed with CellQuest software (Becton Dickinson, Mountain View, CA). In some experiments, non-viable cells were excluded using the vital nucleic acid stain propidium iodide (PI).
For the staining of intracellular Foxp3, cells were incubated with Cy-chrome-labeled anti-CD4 and FITC-labeled anti-CD25 mAbs first [11 (link)]. Then these cells were fixed after washing and stained with anti-mouse Foxp3 mAb, on the basis of the instruction provided by the company (eBioscience, San Diego, CA).