Simultaneous Measurement of Mitophagic Flux and Mitochondrial Morphology During Myogenesis

Mitophagy is the selective degradation of mitochondria via autophagy and is essential for maintaining mitochondrial quality and cellular homeostasis as well as being required for skeletal muscle differentiation and myogenesis. While traditional techniques such as immunoblotting, electron microscopy, and live-cell imaging provide valuable insights, they often offer only static or indirect measurements of mitophagy. Mitophagy flux assays capture dynamic turnover events and are, therefore, more informative. In this chapter, we present a streamlined method for monitoring and measuring mitophagic flux in C2C12 myoblasts during differentiation. Further, this approach allows for simultaneous quantification of mitochondrial morphology, providing a powerful platform for studying mitochondrial quality control in vitro.