Myoblast Fusion Assay for Skeletal Muscle

For the fusion assay of freshly isolated SCs, sorted SCs were cultured for 10 days to induce spontaneous differentiation (Stuelsatz et al., 2015 (link)). For the fusion assay of MPCs, pharyngeal and gastrocnemius MPCs were seeded at low density (5 × 10³ cells/cm²) on Collagen I-coated plates to prevent cell-cell contact and differentiated for 2 days. Then, we counted and seeded cells at high density (7.5 × 10⁴ cells/cm²) to initiate prompt fusion and further differentiated them for additional 2 days. At the end of differentiation, cells were fixed in 2% formaldehyde in PBS for 10 min at room temperature and stained with Phalloidin-iFluor 594 (ab176757; Abcam, United Kingdom) for 30 min at room temperature. Nuclei were then stained with 4′,6-diamidino-2-phenylindole (DAPI), and cells were mounted with Vectashield (Vector Labs, Burlingame, CA). Myoblast fusion was quantified by counting the number of myonuclei in myotubes, which were defined as containing two or more nuclei. Fusion index was calculated as the percentage of myonuclear number relative to the total number of nuclei in the images. Diameters of each myotube were measured at three points (1/4, 2/4, and 3/4 of the length) of a myotube and averaged for each myotube. We collected 10 images from random fields of view for each line.