Enrichment of NK cells from splenocytes using MACS

Splenocytes were isolated as described above. Cells were centrifuged for 5 min at 4°C and 1,500 rpm and cells were resuspended in PBS containing 1 mM EDTA and 2% FBS at a concentration of 1 × 10⁸ cells/ml. Magnetic enrichment of NK cells was achieved by negative selection using EasySep mouse NK cell isolation kit (Cat# 19855, Stemcell Technologies). Briefly, 50 µl/ml EasySep mouse NK cell isolation cocktail was added to the cell suspension and incubated for 10 min at room temperature. EasySep Streptavidin RapidSpheres were vortexed for 30s and 100 µl/ml RapidSpheres were added to the cell suspension and incubated for 5 min at room temperature. PBS/2% FBS/1 mM EDTA was then added to bring the total volume to 2.5 ml and placed into the magnet. After 5 min of incubation, the magnet holding the tube was inverted and the NK cell-containing fraction was collected. The remaining cells were again resuspended in 2.5 ml PBS/2% FBS/1 mM EDTA to collect the remaining NK cells and placed into the magnet for 5 min and the NK cells containing fraction was again collected and combined with the first NK cell fraction. Enriched NK cells were then centrifuged at 4°C for 5 min at 1,500 rpm, supernatant was carefully removed and cells were resuspended in DMEM media supplemented with 10% FBS, 50 U/ml Penicillin, and 50 µg/ml Streptomycin. Purity of MACS-enriched NK cells was then confirmed by flow cytometry.