Isolation and Characterization of Satellite Cells

Satellite cells were isolated from hindlimb and forelimb muscles as previously described (Eliazer et al., 2019 (link)). The mononuclear muscle cells were stained for PE-Cy7 anti-mouse CD31 (clone 390; BD Biosciences), PE-Cy7 anti-mouse CD45 (clone 30-F11; BD Biosciences), APC-Cy7 anti-mouse Sca1 (clone D7; BD Biosciences), PE anti-mouse CD106/VCAM-1 (Invitrogen), and APC anti-α7 integrin (clone R2F2; AbLab). Fluorescence-assisted cell sorting (FACS) was performed using FACS Aria II (BD Biosciences) by gating for CD31⁻/CD45⁻/Sca1⁻/α7 integrin⁺/VCAM1⁺ to isolate SCs. SCs from Pax7-nGFP mouse were sorted for GFP fluorescence. The GFP⁺ gate was divided into top 15% (GFP^high fraction), middle 45% (Pax7^medium fraction), and bottom 15% (Pax7^low fraction). The isolated SCs were fixed immediately at t0 or cultured in growth media (Ham’s F10 media, 20% fetal bovine serum, 5 ng/ml FGF2) containing 10 μm EdU for 60 hr (cell cycle entry assay) or cultured in plating media (DMEM with 10% horse serum) for 3 days (differentiation assay).