Primary Myoblast Isolation and Culture

Adult primary myoblasts were obtained from hind limb muscles of 10- to 20-week-old C57BL/6J mice. Briefly, dissected muscle was digested with collagenase type I (Worthington, USA) for approximately 1 h. A single cell suspension was obtained after filtering the samples through a 70 μm mesh (Falcon). Cells were plated on collagen-coated dishes and cultured for the indicated times in F12-C supplemented with 15% horse serum and 1 nM FGF-2, at 37°C and 5% CO₂ (Olguin and Olwin, 2004 (link); Olguin et al., 2007 (link)). Differentiation was induced by incubating the cultures in F12-C supplemented with 2% horse serum. When required, the inhibitor picropodophyllin (PPP) (Tocris Bioscience, USA; 1 µM) was added for 1 h before treatment with Ang-(1-7).