Mitophagy, a crucial mitochondrial quality control system for cellular stress adaptation, is a key focus in pathophysiology and drug discovery. Developing a simple and versatile mitophagy flux assay is vital for advancing our understanding of cellular responses. Addressing a gap in systematic methods, we employ the photoactivatable fluorescent protein mito-Kaede in C2C12 myocytes, demonstrating its remarkable versatility in quantifying mitophagy flux responses under various stimuli, including carbonyl cyanide m-chlorophenyl hydrazone (CCCP), TNF-α, lipopolysaccharide (LPS), and hypoxia. This study underscores the validity and distinctive advantages of the mito-Kaede assay through comparative analysis with conventional assays including Western blotting (WB), potentially providing valuable insights for both mitophagy flux analysis and drug development.
Keywords: Autophagy; flux measurement; mito-Kaede; mitochondria; mitophagy; quantitative assay; stimulated response kinetics; time-lapse.
The expression of mito-Kaede, which is suitable for monitoring mitochondrial kinetics, was successfully confirmed in the new C2C12 cell line. Mitophagy flux kinetics were measured under four different inducing conditions: CCCP, TNF-α, LPS, and hypoxia. These results were then compared to those obtained from a Western blotting flux-blockade experiment. Additionally, the mito-Kaede assay was evaluated against the mito-QC cell reporter assay. The findings suggested that mito-Kaede provided a more quantitative analysis.