Magnisort mouse nk cell enrichment kit

NK cell cytotoxicity was tested in vitro using 1 × 10⁴ carboxyfluorescein succinimidyl ester (CFSE)-stained YAC-1 or FBL-3 tumor cells and 25 × 10⁴ isolated NK cells from the spleens of naive or FV-infected mice. NK cells were isolated using a MagniSort mouse NK cell enrichment kit (eBioscience). The cytotoxic assay was performed in 96-well U-bottom plates. The cells were coincubated for 24 h in a humidified 5% CO₂ atmosphere at 37°C. Cells were washed once and stained with fixable viability dye (eBioscience) to exclude dead cells. After a washing, cells were directly analyzed by flow cytometry.

Spleens were harvested, and a single-cell suspension was generated following red blood cells lysis and filtration through a 70-µm filter. NK cells were enriched from the spleen by negative selection using the MagniSort Mouse NK cell Enrichment Kit (eBioscience). Purified NK cells were cultured for either 24 or 48 hours with the following cytokines at the indicated concentrations; IL-12 (20 ng/ml), IL-18 (50 ng/ml), and IL-33 (30 ng/ml). 100 U/ml of recombinant human IL-2 (obtained from NCI Preclinical Repository) was added to support NK cell survival. NK cells were cultured in RP-10 media (RPMI-1640 medium containing 10% FBS, penicillin/streptomycin, 2 mM L-glutamine, 10 mmol HEPES, 50 µmol 2-mercaptoethanol). Enriched NK cells were co-cultured with BAF3 or BAF3-m157 cells (gifts from Dr. Wayne Yokoyama, Washington University) at an E:T ratio of 2:1 for 18 hours in the presence of 5 ng/mL IL-15/IL-15Rα complex or 1000U/mL IL-2. Serum levels of IL-18 cytokine were measured using IL-18 Mouse ProcartaPlex™ Simplex Kit (Invitrogen) and acquired using MAGPIX System (Luminex Corporation).