Mouse il 12

One million splenocytes from mixed BM chimeric mice were incubated in 96-well tissue culture plates coated with anti-NKp46 (29A1.4) as described (5 (link)), or with 10 ng/ml mouse IL-12 plus 10 ng/ml mouse IL-18 (R&D Systems), 10 ng/ml PMA plus 1 μg/ml ionomycin (Sigma-Aldrich), or co-cultured with 1 × 10⁵ RMA or m157-transfected RMA cells as described (6 (link)). One million CellTrace Violet-labeled splenocytes were co-cultured with 1 × 10⁵ RMA or m157-transfected RMA cells (fixed in 1% paraformaldehyde) in the absence or presence of 25 ng/ml mouse IL-33 (R&D) and/or 10 ng/ml mouse IL-12 with 50 U/ml human IL-2 (NCI) for 4 days at 37° C.

Splenocytes (1 × 10⁶) from mixed WT and dKO BM chimeric mice were incubated in 96-well tissue culture plates coated with anti-NK1.1 (PK136) or control mouse IgG2a (39 (link)). For cytokine response testing, 1 × 10⁶ splenocytes from mixed WT and dKO BM chimeric mice were incubated with 2.5 ng/ml mouse IL-12 and 2.5 ng/ml mouse IL-18 (R&D Systems). To test degranulation, 1 × 10⁶ splenocytes from mixed WT and dKO BM chimeric mice were co-cultured with either 1 × 10⁵ untransfected or m157-transfected B6 3T3 cells for 5 h at 37° C with PE-conjugated anti-CD107a (clone 1D4B) and GolgiStop (BD Biosciences), followed by staining for surface molecules and intracellular IFN-γ. For assessment of phosphorylated STAT4 and STAT1, 1–3 × 10⁶ splenocytes were cultured with 20 ng/ml mouse IL-12 for 30 min or 1000 U/ml mouse IFN-α (PBL Assay Science) for 10 min, fixed, and stained with AlexaFluor 647-conjugated phosphorylated STAT4 (pY693, clone 38/p-Stat4) or PE-conjugated phosphorylated STAT1 (pY701, clone 4a) (BD Biosciences) (40 (link)).

A total of 2–4 × 10⁴ mouse NK cells were purified and stimulated for 3 h in RPMI plus medium supplement (10% FBS, 1× sodium pyruvate, 1× l-glutamine, 1× pen/strep, 1× MEM-NEAA) with 20 ng ml⁻¹ mouse IL-2 (Thermo Fisher Scientific), 20 ng ml⁻¹ mouse IL-15 (R&D Systems), 20 ng ml⁻¹ mouse IL-12 (R&D Systems), 10 ng ml⁻¹ mouse IL-18 (MBL) and/or 100 IU mouse IFN-α (R&D Systems). Cells were cultured in medium alone as unstimulated controls.