Isolation and Culture of Primary Myoblasts

Primary muscle cells were derived from hindlimb muscles of two-three month-old mice (two mice for each preparation) as described previously^{41 (link)}. Briefly, muscles were digested with 0.2% collagenase type-II (Sigma) in DMEM for 30 minutes at 37 °C, and then with 2 mg/ml Collagenase/Dispase (Roche Diagnostic) for 30 minutes at 37 °C. Satellite cells were mechanically dissociated by passing the tissue suspension through a 5 ml pipette; the slurry was sequentially filtered through 70 and 40 μm cell strainers (BD Biosciences) and centrifuged. Pelleted cells were resuspended in F-10 (Gibco) supplemented with 20% FBS, 2.5 ng/ml basis-FGF (Peprotech), and penicillin-streptomycin. The cell suspension was preplated for one hour on uncoated dishes to remove fibroblasts. The medium containing the enriched myoblast population was then plated on tissue culture dishes coated with type-I collagen (Sigma). After three days in culture, primary myoblasts were shifted to differentiation medium and harvested for RNA preparation after 72 hours.