Mitophagy Quantification via mito-mKeima Biosensor

HeLa and A549 cells were infected with mito-mKeima (m-Keima) expressing lentiviral vector. The fluorescence profile of this biomarker is pH-dependent, making it a perfect biosensor of mitochondrial degradation by the lysosomes [25 (link)]. Excitation at 488 nm and emission at >620 nm was used to detect m-Keima in mitochondria in the cytosol and excitation at 561 nm and emission at >620 nm was used to detect mitochondria in lysosomes (Supplementary Fig. 1A). m-Keima was analyzed by flow cytometry using MACSQuant VYB and MACSQuant software (Miltenyi Biotec, Bergisch Gladbach, Germany).
To calculate the percentage of mitophagy positive cells, 10,000 single events were acquired for each sample and subsequently gated the cells experiencing a shift to acidic m-Keima (Supplementary Fig. 1B).