Detection of CD4+CD25+Foxp3+Tregs and Th cells in spleens and MLNs by flow cytometry
Spleen and MLN lymphocyte single cell suspensions were prepared, and 1×106 cells were added to each tube. The cells were resuspended in PBS and incubated for 30 min in the dark with the following antibody combinations: FITC-rat-anti-mouse CD4, PE-rat-anti-mouse Foxp3 and APC-rat-anti-mouse CD25; FITC-rat-anti-mouse IL-17 and PE-Rat-anti-mouse IL-12; FITC-rat-anti-mouse IL-10 and PE-rat-anti-mouse IFN-γ ; or PE-rat-anti-mouse IL-4 (all antibodies were obtained from BD, USA). The cells were washed and resuspended again for flow cytometry analysis. All data were obtained from three separate experiments.