In Vitro Myoblast Differentiation and Estrogen Interference

C₂C₁₂ cells (ATCC, USA) were cultured in Dulbecco’s modified Eagle’s medium Nutrient Mixture F-12 (DMEM/F12, Hyclone) containing with 10% fetal bovine serum (FBS, Gbico), 100 units/ml penicillin, and 100 μg/ml streptomycin sulfate in a 5% CO₂-humidified chamber (Heraeus, Germany) at 37 °C. For differentiation studies, C₂C₁₂ cells were cultured in DMEM, added with 2% horse serum (Gibco, USA) for 72 h. For proinflammatory stimuli, cells were treated with IFN-γ (0.6 μg/ml, R&D, USA). For in vitro estrogen interference analysis, E₂ (1.0 nM) or 4-OHT (0.1 μM) [34 (link)] were added to the corresponding medium supplemented with IFN-γ, respectively. Cells were analyzed after 48 h culturing with IFN-γ.