Satellite Cell Isolation and Myoblast Differentiation

Satellite cell isolation was performed as previously described, and cell culture was performed similarly to past protocols [9 (link),10 (link)]. Satellite cells were plated on BD Matrigel-coated dishes and activated to differentiate into myoblasts in DMEM-F12, 20% fetal bovine serum (FBS), 40 ng/mL basic fibroblast growth factor (R&D Systems, 233-FB/CF), 1× non-essential amino acids, 0.14 mM β-mercaptoethanol, 1× penicillin/streptomycin, and Fungizone. Myoblasts were maintained with 10 ng/mL basic fibroblast growth factor before they were differentiated in DMEM-F12, 2% FBS, 1× insulin–transferrin–selenium, when 85% confluency was reached. They were cultured at 37 °C, 5% CO₂ Dulbecco’s modified Eagle’s medium (DMEM; GIBCO) supplemented with 10% fetal bovine serum (FBS; Atlanta Bio selected), and antibiotics-1% penicillin-streptomycin (Gibco, Waltham, MA, USA). Three days after differentiation, myotubes were infected with adenovirus for ntGFP or GFP-Cre for OPA1 deletion at a multiplicity of infection sufficient to infect >95% of the cells with minimal cell death. Adenoviruses were obtained from the University of Iowa Viral Vector Core facility. Experiments were performed between 3 and 7 days after infection for a total of 6 days of differentiation.