Isolation of Skeletal Muscle Stem Cells

Hindlimb muscles (quadriceps, gastrocnemius, and tibialis anterior) were dissected and MuSCs collected as described [36 (link)]. Briefly, tissue was enzymatically dissociated with 0.1% collagenase (Sigma) and 4.8 units/mL dispase (Roche) in DMEM, using the gentleMACs system (Miltenyi Biotech). The cell slurry was pulled through a 21-gauge needle until all remaining muscle tissue was broken apart, after which the cell solution was filtered through a 40 μm cell strainer. Red blood cells were eliminated with red cell lysis buffer (eBioscience). Cells were stained with biotinylated antibodies (CD31 from eBioscience; Sca1, CD45, and CD11b from BD Biosciences) followed by staining with streptavidin-conjugated PE-Cy7 (BioLegend), Alexa Fluor 647-conjugated α7-integrin antibody (AbLab), and Brilliant Violet 421-conjugated CD34 antibody (BD Biosciences). The viability dye 7-AAD (Sigma) was added and cells were either sorted on a FACS Aria II cell sorter (BD Biosciences) or analyzed on an LSRII (BD Biosciences).