Mouse maGSCs (line SSC5) were cultured on mitomycin C-inactivated mouse embryonic feeder cells in Dulbecco’s modified eagle medium (DMEM; Thermo Fisher Scientific) supplemented with 15% fetal calf serum (FCS; selected batch, Lonza), glutamine (2 mM, Thermo Fisher Scientific), 1× non-essential amino acids (Thermo Fisher Scientific), β-mercaptoethanol (β-ME; 50 µM, Promega), and 103 U/ml leukemia inhibitor factor (Millipore) as described previously (7 (link)). The cells have been derived from a mouse with a mixed FVB (H2q), C57BL/6 (H2b), and 129Sv (H2b) genetic background (7 (link)). For in vivo studies, the maGSCs were separated from the mouse embryonic feeder cells before use. The maGSCs were obtained by collecting the floating cells after being cultivated for 1 h on culture dishes coated with 0.1% gelatin (Sigma-Aldrich: Fluka).
The murine T-lymphoma cell lines YAC-1 (H2a), which was used as positive control for the cytotoxic activity of NK cells, and RMA (H2b) were maintained in DMEM supplemented with 10% FCS, 2 mM glutamine, 1 mM sodium pyruvate, 50 µM β-ME, 100 U/ml penicillin, and 100 µg/ml streptomycin.
The murine T-lymphoma cell lines YAC-1 (H2a), which was used as positive control for the cytotoxic activity of NK cells, and RMA (H2b) were maintained in DMEM supplemented with 10% FCS, 2 mM glutamine, 1 mM sodium pyruvate, 50 µM β-ME, 100 U/ml penicillin, and 100 µg/ml streptomycin.