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Mouse primary myoblasts were maintained in DMEM media supplemented with 20% fetal bovine serum, 10% horse serum and 1% penicillin/streptomycin. To induce myogenic differentiation 90% confluent cells were cultured in DMEM supplemented with 5% horse serum and 1% penicillin/streptomycin (DM). Myoblast differentiation was assessed 10 days following the switch to DM by immunostaining for myosin heavy chain using the MF20 antibody52. Images were taken using Zeiss fluorescent microscope and analysed using ImageJ as in25.
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