H-2Kb cells were derived from skeletal muscle of heterozygous H-2kb tsA58 transgenic mice. Myoblasts were maintained under permissive conditions in Dulbeccos modified serum containing heat-inactivated fetal calf serum (20% (v/v), chick embryo extract (2% (v/v)), penicillin/streptomytcin (1% (w/v)), and L-glutamine (2% (v/v)) at 33.C in the presence of interferon-gamma to keep in undifferentiated state (20U/ml). The medium was changed twice weekly. Differentiation into myotubes was induced following a switch to non-permissive growth conditions by removal of interferon and incubation at 37.C for four days. Then it was transferred into media containing 0.5% (v/v) fetal calf serum, and incubated overnight before use. The following morning the cells were rinsed twice in Phosphate Buffered Saline (PBS) buffer and then incubated at 37.C in HEPES buffered saline (HBS) containing 5mM glucose in the presence or absence of insulin (100nmoles)/metformin (2mM). Start the time course experiments.

Ref: H2K-b muscle cell lines isolated from H-2Kb-tsA58*mdx/mdx transgenic mice. Jat et al., PNAS: (1991): 88, 5096-5100.

Experiment E-BAIR-7