Il 2 elisa kit

The human IFN-gamma ELISA kit, IL-2 ELISA kit, and TNF-alpha ELISA kit (all kits, Dakewe, Beijing, China) were used to measure the concentrations of IFN-γ and IL-2, TNF-α, respectively. According to the instructions of the ELISA kit, three samples were processed, and the standard curve was prepared. Then the fluorescence value was measured by the enzyme-labeled instrument (TECAN, Mannedorf, Switzerland). The cytokines quantity was then calculated.

SBF-1 was synthesized by Prof. Biao Yu, a coauthor, as previously reported [14 (link)]. SBF-1 was dissolved in DMSO and stored at − 20 °C. For in vitro studies, the stock solution concentration was 20 mM. For in vivo studies, the stock solution concentration was 10 mg/ml. The final DMSO concentration did not exceed 0.1% throughout the study. Concanavalin A (Con A) was purchased from Sigma-Aldrich (St. Louis, MO). Purified anti-mouse CD3 and anti-mouse CD28were purchased from BD Biosciences (San Jose, CA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Annexin V/PI kit were purchased from Sunshine Biotechnology (Nanjing, China). Fetal bovine serum (FBS), RPMI 1640, penicillin and streptomycin were purchased from Life Technology (Carlsbad, CA). FITC anti-mouse CD25 antibody and FITC anti-mouse CD69 antibody were purchased from eBioscience (San Diego, CA). IFN-γ ELISA kit and IL-2 ELISA kit were purchased from Dakewe Biotech (Beijing, China). Primary antibodies against AKT (pS473) and AKT were purchased from Cell Signal Technology (Beverly, MA). Antibody against GAPDH was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Picryl chloride (PCl) was purchased from Wako Pure Chemical Industries (Osaka, Japan).