Anti mfoxp3 pe

Treg cell analyses were performed by flow cytometry using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). The spleens were removed aseptically and mechanically dissociated in the presence of DMEM medium. The dissociated cells were centrifuged and the pellet was resuspended in hemolytic solution, followed by additional centrifugation. The pellet was resuspended in DMEM medium supplemented with 5% fetal bovine serum and the numbers of splenocytes were counted using a Neubauer chamber. Erythrocyte-depleted spleen cells (2 × 10⁶ cells) were incubated with anti-CD4-FITC (BD Pharmingen) and anti-CD25-APC (BD Pharmingen) at 4 °C for 30 minutes, followed by washing with FACS Buffer (PBS, 0.5% BSA and 0.01% NaN₃). After this, cells were fixed and permeabilized using the Mouse Regulatory T-Cell Staining Kit (eBioscience) and stained with anti-mFoxP3-PE (eBiosciences), according to the manufacturer’s instructions. The data were analyzed using cellquest™ Pro Software (BD Biosciences).

The monoclonal antibodies anti-CD3-PE, anti-CD4-APC, and anti-CD25-FITC and all isotype controls were purchased from BD Biosciences-Pharmingen, and anti-mFoxp3-PE was obtained from eBioscience. We used FACSCalibur (BD) and CELLQuest software (version 3.3; BD Biosciences-Pharmingen) to analyze lymphocyte surface markers. Briefly, 50–100 μl of blood was collected from mice in heparin-coated tubes, and erythrocytes were lysed using standard protocols. The remaining lymphocytes were washed three times with PBS and incubated with anti-mFoxp3-PE and anti-CD4-APCs and subsequently analyzed on a FACSCalibur (BD) flow cytometer with CELLQuest software (version 3.3; BD Biosciences-Pharmingen) as previously published [15 (link)].