Duoset human il 2 kit

Jurkat T-CD16A cells were used to evaluate the engagement of CD16A on the effector cell surface by the BiKEs through monitoring of the IL-2 secretion. Jurkat T-CD16A cells were cultured in RPMI1640 medium (Invitrogen) containing 10% FBS, 1 mM pyruvate, 0.1 mM MEM non-essential amino acid, 250 μg/mL G418 and 100 μg/mL hygromycin (Invitrogen). Before incubation with BiKEs, Jurkat T-CD16A cells were activated overnight by Phorbol-12-myristate-13-acetate (PMA) (50 ng/ml, Sigma). Then 10⁶ Jurkat T-CD16A cells were incubated with BiKEs (20 nM) alone or with BiKEs/mouse anti-Flag mAb (1: 1000 dilution) mixture or with BiKEs opsonized target cells, 293T-gp160_SC cells. The mouse anti-CD16A IgG1, 3G8, was used as a positive control. After incubation, cells were centrifuged for 10 min at 1200 × g and the quantity of human IL-2 in the supernatant was measured by sandwich ELISA with the Duoset Human IL-2 kit (Duoset Human IL-2, R&D Systems) according to the manufacturer’s instructions.