C2C12 Myoblast Differentiation and Atrophy

C2C12 cells (mouse skeletal myoblasts) were obtained from ATCC and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Corning, NYC, USA, 10-013-CV) with 10% Fetal bovine serum (Biological Industries, Beit HaEmek, Israel) and 1% Penicillin-Streptomycin (KeyGEN, Nanjing, China, KGY0023) at 37 ℃ supplemented with 5% CO₂. To differentiate into myotubes, C2C12 myoblasts cells were cultured in a differentiation medium (DMEM containing 2% horse serum and 1% penicillin and streptomycin) and the entire differentiation process lasted about 4 days. EV-depleted Fetal bovine serum (FBS) was obtained after overnight centrifugation at 100,000 g at 4 °C (Beckman Coulter, Avanti JXN-30). For screening the optimum concentration, EVs were added to the medium for 24 h at different doses. For determining the function of EVs in muscle atrophy in vitro, EVs were added to the medium at a dose of 1 × 10¹⁰ particles/mL for 24 h, then muscle atrophy was induced. Dexamethasone (Dex, Sigma, MO, USA, D4902), TNF-α (PeproTech, NJ, USA, 315-01 A), and Ang II (Sigma, MO, USA, A9525) were used to induce muscle atrophy at the cellular level according to previously reported methods [4 (link)].