Recombinant human il2 rhil2

Human PBMCs were isolated by Ficoll-Paque PLUS (GE Healthcare) density gradient centrifugation, washed twice in PBS, and resuspended at 106/mL in complete RPMI 1640 (cRPMI) medium containing 10% FBS, 2 mM glutamine, and 100 U/mL each of penicillin and streptomycin (Invitrogen). Cells were activated with 1 µg/mL anti-CD3 (clone HIT3α, BioLegend) and 1 µg/mL anti-CD28 (clone CD28.2, BioLegend). After 3 days, activated T cells were washed and then cultured in cRPMI with 100 U/mL Recombinant Human IL-2 (rhIL-2, R&D). In the case of patient cells, freshly isolated or frozen PBMCs were stimulated at 5x106/mL in cRPMI with 2 µg/mL of PHA-L (lectin from red kidney bean (Phaseolus vulgaris), Sigma-Aldrich L2769), in the presence of 1 µg/mL anti-CD28 and 1 ng/mL recombinant IL7 (BioLegend) for 48 hours. The cells were washed twice with cRPMI and resuspended in media at 1 x 106/mL with 100 IU/mL Recombinant Human IL2 and cultured for up to 3 weeks with fresh rhIL2 and medium supplemented every 2 days.