Cd3 depletion kit

Human iPS cells (Thermo Fisher) were cultured on vitronectin-coated T225cm² flasks using complete mTesSR Plus medium (StemCell Technologies) supplemented with 1% penicillin/streptomycin, Rock inhibitor (StemCell Technologies) at 1:1000 dilution. For drug selection, G148 was used at 500ug/mL and puromycin at 5ug/mL (Sigma-Aldrich). Cultures were maintained at 37°C, 5% CO2 in a humidified incubator. NK cell effectors were enriched from normal cynomolgus macaque (Macaca fascicularis, male and female) blood samples using a CD3 depletion Kit (Miltenyi Biotec). NK cells were maintained in RPMI 1640 with Glutamax (Life Technologies) supplemented with 10% heat-inactivated FCS, 2 mM, l-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin and 100 IU/mL of IL-2 and 10 ng/mL of IL-15.

Both MHC-I expressing and MHC-I deficient (B2M knockout) UVC were used as target cells for NK cell cytotoxicity assay. Trypsinized cells were stained with calcein acetoxymethyl ester (CAM, Invitrogen) at a 10 μM concentration for 1 h at 37°C and then washed to remove excess dye. NK cells highly enriched from normal cynomolgus macaque (Macaca fascicularis) blood samples using a CD3 depletion kit (Miltenyi Biotec), were used as effector cells. NK cell effectors and stained target cells were co-cultured in 96 well round bottom plates at effector: target (E:T) ratios of 1:1 and 5:1. Control wells included – only target cells for spontaneous release of CAM and target cells treated with Triton X-100 for maximum release of CAM. At the end of 4-h incubation, supernatant was collected for CAM measurement in a fluorescent plate reader at 530 nm. Percent-specific lysis = (test release - spontaneous release)/(maximum release - spontaneous release).