Isolation and Culture of Murine Satellite Cells

MuSCs were isolated as previously described^{7 (link),11 (link)}. Briefly, TA muscles from 1-month-old mice were dissected and dissociated with collagenase (Roche). The cells were negatively selected by biotinylated CD45, CD11, CD31 and Sca1 antibodies. The muscle cells in the flow-through were subjected to CD34-FITC (BD Biosciences) and integrin-α7-allophycocyanin (R&D systems) staining. The viable PI⁻CD34⁺ integrin-α7⁺ MuSCs were collected by FACS sorting. MuSCs were cultured on collagen-coated dishes in F10 basal medium (F10 medium containing 15% FBS and 2.5 ng/ml FGF (Invitrogen)), T cell conditional medium (F10 medium with 10% FBS:T cell medium = 50:50) or cytokine cocktail medium (F10 medium containing 10% FBS, 5 ng/ml IL-1α, 5 ng/ml IL-13, 10 ng/ml IFN-γ and 10 ng/ml TNF-α (R&D Systems), and 2.5 ng/ml FGF). For serial expansion, 10 000 cells were seeded in a 3.5-cm dish, and the cells were expanded every 2 days. MuSCs were differentiated in differentiation medium (DMEM (Invitrogen) with 2% horse serum (Sigma)).