For B-cell differentiation, isolated naive B cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 supplemented with 10% fetal bovine serum (FBS) (Life Technologies) and 1% penicillin/streptomycin (Life Technologies). Cells were stimulated with antihuman IgM (5 μg/ml, Sigma, #10759) and antihuman CD40 (1 μg/ml, Bioxcell, #BE0189) antibodies in the presence of interleukin (IL)-2 (20 ng/ml, PeproTech), IL-4 (25 ng/ml, Sino Biological) and IL-21 (50 ng/ml, Sino Biological) for 8 days.
For signaling study, B cells were stimulated with antihuman IgM (5 μg/ml) and antihuman CD40 (1 μg/ml) antibodies for 72 h. CRAC channel inhibitor YM-58483 (50 nM, MedChemExpress, USA) or CaMK2 inhibitor KN-93 (5 μM, Selleck) was included in some of the experiments. Cells were maintained in a humidified atmosphere at 37°C with 5% CO2. Cells were collected for Western blot or FACS analysis.
For signaling study, B cells were stimulated with antihuman IgM (5 μg/ml) and antihuman CD40 (1 μg/ml) antibodies for 72 h. CRAC channel inhibitor YM-58483 (50 nM, MedChemExpress, USA) or CaMK2 inhibitor KN-93 (5 μM, Selleck) was included in some of the experiments. Cells were maintained in a humidified atmosphere at 37°C with 5% CO2. Cells were collected for Western blot or FACS analysis.