Percp cy 5.5 mouse anti human cd25

Samples were washed with PBS and fixed with immobilization reagent (BD Biosciences). Type 17 T-helper (Th17) cells were stained with allophycocyanine mouse anti-human CD4 (cat. no. 561840; BD Biosciences) for 20 min at 37˚C, phycoerythrin (PE) mouse anti-human interleukin (IL)-17A (cat. no. 580487; BD Biosciences) for 30 min at 4˚C, PerCP-Cy™ 5.5 mouse anti-human CD25 (cat. no. 580503; BD Biosciences) for 20 min at 4˚C, PE mouse anti-human CD127 (cat. no. 561028; BD Biosciences) for for 20 min at 4˚C. A total of 1x10⁶ of cells were resuspended in 1% BSA-PBS and the volume of fluorescent antibodies added in to cells were as listed below: CD4: 1 µl; IL-17A: 2 µl; CD25: 2 µl and CD127: 2 µl. After incubation with the antibodies, cells were analyzed using flow cytometry on a BD FACSCalibur (BD Biosciences).
ELISA kits (cat. nos. m1037279 and m1028599; Shanghai Enzyme Link) were used to detect the levels of IL-17 and transforming growth factor (TGF)-β in human serum (1:5 dilution), according to the manufacturer's instructions.