T cell activation from PBMCs

To activate and expand T cells from PBMCs, thawed PBMCs from patient, patient’s father, or healthy donor resuspended in 10% complete RPMI 1640 medium [18 (link)] were stimulated with 1 μg/ml of anti-CD3 (clone HIT3a, BD Pharmingen 555336) and anti-CD28 (clone CD28.2, BD Pharmingen 555725) antibodies, and plated at 2 × 10⁶ cells per well of 48-well plate. PMBCs were incubated at 37 °C and under 5% CO₂ for overnight and then continue cultured in the presence of 30 U/ml hIL-2 (PeproTech) in medium for 7 days. Fresh hIL-2 containing medium was added every 2 days. Orai1-KO HEK293 [19 (link)] cells were in DMEM media (Invitrogen) supplemented with 10% FBS (Gibco), 100 U/ml penicillin, and 100 μg/ml streptomycin. Transient transfections were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. The transfection efficiency was routinely checked by low magnification microscopy on an EVOS FL Cell Imaging System (ThermoFisher Scientific). For 4-PBA treatment, cells were first transfected with plasmids for 6 h and then treated with 10 μM of 4-PBA for additional 12 h.