Foetal and adult muscles were dissected and minced in ice-cold DMEM as described in [39]. Samples were then incubated in DMEM, 0.08% Collagenase D (Sigma, 11088882001), 0.2% Trypsin (ThermoFisher, 15090) and 10 μg/ml of DNAseI (Sigma, 11284932) for 25 min at 37 °C under gentle agitation for 5 rounds of digestion. After each round, samples were allowed to sediment for 5 min, the supernatant was collected in 4 ml of foetal bovine serum (FBS) on ice and fresh digestion buffer was added to the remaining muscle pellet. The collected supernatants were centrifuged for 15 min at 550g at 4 °C, resuspended in DMEM 2% FBS, and filtered through a 40-μm strainer (Corning, 352235) before cell sorting. Cells were isolated based on size, granulosity and GFP or tdTOM fluorescence using an Aria III (BD Biosciences) flow cytometer. Cells were collected directly in MuSC growth media (38.5% DMEM (Fisher Scientific, 31966047), 38.5% F12 (Fisher Scientific, 31765035), 20% FBS (ThermoFisher, 10270), 2% Ultroser (Pall, 15950-017), 1% penicillin/streptomycin (GIBCO, 15140-122)).

Matrigel® (1 mg/ml, Corning, 354248) coated dishes (30 min at 37 °C) were used to culture MuSCs in growth media at 3% O2, 5% CO2, 37 °C for the indicated times.

For immunostaining, cells were fixed in 4% paraformaldehyde (PFA, Electron Microscopy Sciences, 15710) in PBS for 15 min at room temperature (RT), permeabilised in 0.5% Triton X-100 (Merck, T8787) for 5 min at RT and blocked with 10% goat serum (GIBCO). Cells were incubated with the indicated primary antibodies in PBS 2% goat serum buffer overnight following by 45-min incubation with secondary antibodies and 1 μg/ml Hoechst (ThermoFisher, H1399).

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