Quantifying C2C12 Myoblast Fusion

For measuring fusion, C2C12 myoblasts were washed and fixed with 4% paraformaldehyde for 20 min and then permeabilized in 0.1% Triton X-100 for 10 min. After blocking with goat serum (Boster, China), C2C12 myoblasts were incubated with mouse anti-myosin antibody (R&D, MAB4470) at 1:250 dilution overnight at 4 °C and then incubated with Alexa Fluor 594 labeled secondary antibody (Yeasen, 33212ES60) at 1:500 dilution for 1 h at room temperature and stained with Hoechst 33342 (Invitrogen, H21492) for 5 min at room temperature. The fluorescently stained C2C12 myoblasts were captured by Operetta CLS (PerkinElmer, USA) for analysis. The fusion index and the number of multinuclear myotubes were quantitated based on myosin⁺ cells per field by Image J software referred to the previous study^{34 (link)}.