Flow Cytometry Detection of Treg Cells

Blood samples (300 ul) collected from AA mice and normal controls at day 30 were processed with red blood cell lysate. The cells isolated from PBMCs were stained with allophycocyanin (APC)- Cyanine7 -anti-mouse CD4, PE-Cyanine 7-anti-mouse CD8, APC-anti-human CD25 for 25 min at 4°C. Afterwards, fixation and permebilization of the cells were performed for Foxp3 staining as an intracellular marker. The fixed and permebilized cells were stained with PE-anti-mouse Foxp3 antibody for 25 min at 4°C. The cells were washed twice with 1 ml PBS and centrifuged at 300 × g for 5 min at room temperature. The percentage of the stained cells was measured by a FACSanto II flow cytometer (Becton Dickinson, San Jose, CA). The CD4⁺, CD25⁺, and Foxp3⁺ cells were considered as Tregs. The monoclonal antibodies related were bought from eBioscience, USA.