Activation of Frozen Immune Cells

Liquid-nitrogen frozen PBMC from healthy donors and from T1D patients were thawed in complete RPMI medium (Gibco RPMI 1640 Medium, ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Hyclone, South Logan, UT, USA), L-glutamine (2mM) (EuroClone S.p.A., MI, Italy) and 1% penicillin/streptomycin (pen/strep) (EuroClone) according to established protocols [19 (link)] and centrifuged at 1200 rpm for 5 minutes at room temperature (RT). Cells were cultured in 48 well plates (Falcon, Corning Incorporated, NY, USA), 1.5×10⁶ cells per well in complete RPMI. Subsequently, cells were stimulated with the addition of 7.5 ng/ml phorbol-12-myristate-13-acetate (PMA) (Calbiochem, Merk, Darmstadt, Germany) and 0.8μg/ml Ionomycin (IONO) (Sigma Aldrich, Merk). The cells were incubated for three to five days at 37°C in a humidified atmosphere containing 5% CO₂. At the end of the incubation period, cells were harvested from culture plates and washed by centrifugation 1200 rpm for 5 minutes in PBS at RT. Subsequently cells were stained for FACS analysis as described below.