Monitoring Mitophagy Dynamics in Live Cells.

The purpose of this protocol is to provide a comprehensive, stepwise guide for assessing mitophagy flux utilizing a live-cell mt-KEIMA approach. The proposed protocol is sensitive, reproducible, quantitative, and easy to perform. While mitophagy has been extensively studied, current methodologies primarily focus on terminal measurements, neglecting the dynamic aspect of this process. Hence, the introduction of this straightforward live-cell mitophagy tracing protocol enables real-time monitoring of the dynamics of mitochondrial selective autophagy, thereby enhancing the ability to draw conclusions regarding key regulators and the reversibility of the process. The assay employs a lentiviral approach to induce mt-KEIMA expression in primary or immortalized cell lines. Subsequently, the respective mitophagy reporter cells are observed using a live-cell imaging system at specific time intervals, and further quantification allows the detection of mitophagy flux. This protocol has proven efficacious in investigating mitophagy flux, including responses to chemical inducers or genetically modified cells over time. Notably, this approach is well-suited for large throughput screening of chemicals or appropriate gene-editing libraries that may influence mitophagy responses in cells.