Myogenic Differentiation Induction and Characterization

When the cultured cells reached 80-90% confluence, myogenic differentiation was induced by low serum medium comprised of DMEM supplemented with 2% horse serum (Hyclone Ltd) for 5 days. Myogenic cells and myotubes were identified by immunostaining, as described previously 37 (link). Myogenic cells were incubated with primary antibodies against MyoD (1:200; Santa Cruz, USA) and myotubes were incubated with primary antibodies against MHC (1:200; Cell Signaling Technology, USA) at 4℃ overnight, then incubated with fluorescence-tagged secondary antibodies (1:500; Santa Cruz, USA) at 37℃ for 1 h. After incubation and mounting, the samples were stained with 4',6-diamidino-2-phenylindole (DAPI) for 20 min. Samples were observed and photographed using a confocal fluorescence microscope (A1R/A1; Nikon, Japan).