Isolation and Culture of Satellite Cells

Satellite cells were sorted based on established methods^{81 (link),82 (link)}. Briefly, entire hindlimb muscles from mice were digested with collagenase II (LS004177, Worthington, 1000 units per 1 ml) for 90 min at 37 °C, the digested muscles were then washed in washing medium (Ham’s F-10 medium (N6635, Sigma) containing 10% horse serum, heat-inactivated (HIHS, 26050088, Gibco, 1% P/S) before SCs were liberated by treating with Collagenase II (100 units per 1 ml) and Dispase (17105-041, Gibco, 1.1 unit per 1 ml) for 30 min. The suspensions were passed through a 20G needle to release myofiber-associated SCs. Mononuclear cells were filtered with a 40-µm cell strainer and sorted by BD FACSAria IV (fluorescence-activated cell sorting) with the selection of the positive GFP fluorescence signal. BD FACSVerse flow cytometer, BD FACSAria Fusion Cell Sorter and BD FACSDiva (Version 8.0.1, BD Biosciences) were used for the acquisition of flow cytometry data. Coverslips and cultural wells were coated with poly-D-lysine solution (p0899, Sigma) at 37 °C for overnight and then coated with extracellular matrix (ECM) (E-1270, Sigma) at 4 °C for at least 6 h. FACS-isolated SCs were seeded in coated wells and cultured in Ham’s F10 medium with 10% HIHS, 5 ng ml⁻¹ β-FGF (PHG0026, Thermo Fisher Scientific) and 1% P/S.