2.6. Percentage of CD4+CD25+ and Foxp3+CD4+CD25+ Treg Cells in Rats Spleen Lymphocytes Determined by Flow Cytometry

The spleens were cut into pieces, washed with RPMI-1640 culture medium, and centrifuged for 5 min at 1500r/min. After filtering with 100-mesh nylon mesh, the supernatant was discarded, and the spleen cells of each group were suspended with RPMI-1640 culture medium, and then rat spleen lymphocytes were isolated and collected by using a rat peripheral blood lymphocyte separation medium kit.

Isolated rat spleen lymphocytes were washed twice with PBS containing 1% fetal calf serum (FCS) and then resuspended in it at a concentration of 1 x 10⁶ cells/ml. One hundred microliters of the solution of each group was transferred into a 5 mL culture tube; then 5 μl of PE Mouse anti-rat CD4 mAb and FITC Mouse anti-rat CD25 mAb was added. The cells were gently vortexed and incubated for 30 min at room temperature in the dark. Next, 400 μl of PBS containing 1% FCS was added to each tube. The prepared samples were analyzed by flow cytometry (Beckman Cytomics FC 500) within 1 hour.