Recombinant human IL-38 protein stimulated PBMCs

PBMCs were counted and spread in 12-well sterile cell culture plates at a density of 1×106/ml/well in each group and cultured in 5 groups using RPMI 1640 (containing 10% fetal bovine serum). Anti-CD3 and Anti-CD28 antibodies at a final mass concentration of 1 μg/ml were added to each group to promote the activation of T-lymphocytes. The final concentrations of recombinant IL-38 protein in each group were set at 0, 100, 200, 500, and 1000 ng/ml. The IL-38 concentration of 0ng/ml was considered a blank control group. The cell culture plates were incubated in a sterile cell culture incubator at 37 °C, 5% CO2 for 48 h, and then the cells were collected (using a high-speed frozen centrifuge at 4 °C, 1000 ×g for 20 minutes). The cells were then resuspended using RPMI1640 culture medium, and their cell concentration was adjusted to 1×107/ml. From each sample, 250 μl of PBMCs was removed from the resuspension into a clean test tube. The percentage of CD4+ T cell sub-populations in each set of PBMCs was then analyzed using the previously outlined flow cytometry approach.