Mitocapture

A reduction in mitochondrial membrane potential is an early indicator of apoptosis induction (Yano et al., 2020 (link)). The membrane potential was measured using the MitoCapture mitochondrial apoptosis kit (BioVision, Milpitas, CA, United States ) according to the instructions as described. Briefly, once the C2C12 myoblasts were subjected to the treatments as outlined in the protocol above, they were incubated in the cultured medium containing the MitoCapture reagent at a 1:1,000 dilution for 20 min at 37°C. Following two washes with PBS, fluorescent signals were measured using a confocal laser scanning microscopy (LSM 700, Carl Zeiss). The signal for capturing red fluorescence was excited to 530 nm and detected at 630 nm; the signal for capturing green fluorescence was excited to 488 nm and detected at 530 nm. The intensity for quantifying fluorescent signal was assessed by using NIH Image J software.
TMRM assay. Once C2C12 myoblasts were subjected to different treatment as outlined above, they were incubated in serum free medium containing TMRM at a concentration of 300 nM (Thermo Fisher) for 30 min at 37°C in the presence and absence of pre-treatment with FCCP (20 µM) for 20 min. The fluorescent signals were acquired at excitation to 547 nm and detection at 573 nm. Intensity of fluorescent signals was evaluated through NIH Image J imaging software.