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Fig. 1. Myotube formation of C2C12 myoblasts through three distinct stages. (A) Differentiation of C2C12 cells was induced in a serum-free DMEM containing 10 ng/ml IGF-I. Along with typical morphological changes, the expression levels of PCNA and MHC were examined by immunostaining, using specific antibodies. There was no overlapping of the staining for proliferation and differentiation markers, indicating that there were three distinct stages, as shown on the right. These stages were designated as a proliferation stage, an elongation stage and a fusion stage, based on their cell morphologies. Bars, 100 µm. (B) Cell and myotube nuclei were counted. A fusion ratio was calculated as nuclei in multinucleated cells/total nuclei. An increase in the number of nuclei in the early phase (24 hours), and the fusion index increase in the late phase (72, 96, 120 and 144 hours) clearly established the boundaries between the proliferation and elongation stages at 24 hours, and between the elongation and fusion stages at 72 hours.
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