Isolation and Differentiation of Primary Myoblasts

Primary myoblasts were isolated from hind limb skeletal muscles of Mfn2^MKO and control mice at 6-week old.^{33 (link)} Muscles were minced and then digested in 0.1% type I collagenase and 0.24% Dispase B mixture (Roche Applied Science). The digestions were stopped by adding F-10 Ham's medium (Thermo Fisher Scientific) containing 20% fetal bovine serum (FBS, Atlanta). Cells were then filtered through 70-μm filter to remove debris, centrifuged at 1700 rpm (Thermo Fisher CL2) for 5 minutes, and cultured in F-10 Ham's medium supplemented with 20% FBS, 4 ng/mL basic fibroblast growth factor (bFGF, Thermo Fisher Scientific), and 1% penicillin–streptomycin (Thermo Fisher Scientific) on collagen-coated cell culture plates at 37°C, 5% CO₂. For differentiation, primary myoblasts were plated on Matrigel (Corning No. 354234) coated cell culture plates at 70% confluence. Myoblasts were induced to differentiate in differentiation media: DMEM (Thermo Fisher Scientific) supplemented with 2% horse serum and 1% penicillin–streptomycin.