Foxp3 staining for flow cytometry detection

A Foxp3 staining kit (Ebioscience, San Diego, CA, USA) was used for Foxp3 staining. Briefly, we added 1 mL of 1× Foxp3 Fix/Perm solution to resuspended cell pellets and incubated the mixture at room temperature in the dark for 20 minutes. After washing and resuspending the cell pellets in 1× Foxp3 Perm buffer, the cells were added to an appropriate amount of PE-conjugated anti-human Foxp3 antibody and incubated at room temperature in the dark for 30 minutes. We washed the samples twice with cell staining buffer and resuspended them in 300 µL of PBS for flow cytometry. The CD3⁺CD4⁺Foxp3⁺ strategy was used to calculate the proportion of Tregs.