Isolation and Preservation of Murine Muscle Stem Cells

Single myofibers were isolated from extensor digitorum longus (EDL) muscles as described (Li and Fan, 2017 (link)). Isolated MuSCs were fixed in 4% PFA immediately or cultured in DMEM with 10% horse serum and 0.5% chick embryo extract for specified time in text, figures, and legends. To preserve MuSC projections, modifications were made to the procedure in (Kann et al., 2022 (link)). Knee tendon was cut prior to ankle tendon to remove the EDL muscle. Tugging and pulling were avoided to prevent muscle stretching and loss of projections. EDL muscles were digested in 2.6 mg/mL collagenase in DMEM with Y-27632 (50 μM; Tocris Bioscience) for 55 min in 37°C shaking water bath, and transferred to DMEM with Y-27632 (50 μM) for trituration to liberate individual myofibers. These single myofibers were immediately fixed in 4% PFA. After fixation, myofibers and their associated MuSCs were subjected IF and imaging analysis.