Murine Skeletal Muscle Cell Differentiation

Murine skeletal muscle cell line (C2C12) was purchased from American Type Culture Collection (Manassas, VA, USA). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)) supplemented with 20% fetal bovine serum (FBS) and 100 U/mL penicillin/streptomycin (Thermo Scientific, Waltham, MA, USA). Cells were maintained at 37 °C in humidified air containing 5% CO₂. To induce C2C12 differentiation, cells were plated in growth medium, which was replaced by differentiation medium composed by DMEM supplemented with 2% heat-inactivated horse serum (Biowest, Nuaillé France) and 100 U/mL penicillin/streptomycin after 48 h and maintained for 3 days [16 (link)].