Stimulation of NK cell cytokine production

NK cells were prepared from bone marrow or splenocytes of RAG^−/− mice (B6.12957-RAG1tm1Mom, Jackson). Bone marrow from RAG^−/− mice was cultured in complete media in the presence of recombinant human IL-2 (Proleukin) for seven days. On day 7, NK cells were harvested, plated at 3×10⁵ cells/well and stimulated with 0, 0.1, 0.5, 1, 5 or 10 ng/ml IL-12 (eBioscience). At each concentration of IL-12, NK cells were either stimulated with 50 U/ml TNF-α (eBioscience), 10 ng/ml Flt3L (eBioscience), 100 ng/ml Flt3L, or left unstimulated (37 (link), 38 (link)). Cells were incubated for 48 hours and IFN-γ production was measured by ELISA. Splenocytes from RAG^−/− mice were cultured for 48 hours under the conditions described above, and expression of CD69 and KLRG1 was measured on NK cells by flow cytometry.