C2C12 mouse myoblast cell culture and differentiation into myotubes

Frozen murine C2C12 myoblasts (2 × 10⁶ cells) were thawed in a 37°C water bath and then seeded in flasks (130191; Thermo Fisher) containing 20 ml of GM, which consisted of DMEM base media (10013CV; Corning), 10% FBS (SH30071.03; GE Healthcare Life Sciences), and 0.1% penicillin and streptomycin (15140122; Thermo Fisher) and maintained in a humidified incubator at 37°C with 5% CO₂ until 70% confluent (∼2 d). The GM was aspirated, the cells were washed with PBS, and adherent cells were dislodged using 3 ml of 0.25% trypsin (25053CL; Corning). Cells were then seeded 0.3 × 10⁶ cells per well in 6-well plates (140685; Thermo Fisher) with GM and maintained at 37°C with 5% CO₂ until 90% confluent. The GM was then aspirated, cells were washed with PBS, and 2 ml per well of differentiation media (DM) consisting of DMEM base media (10013CV; Corning), 2% horse serum (26050–088; Life Technologies), and 0.1% penicillin and streptomycin (15140122; Gibco) was added and cells maintained at 37°C with 5% CO₂. The DM was replaced every 48 h, and C2C12 myoblasts were fully differentiated into myotubes after 5–6 d in DM.