Mitochondrial autophagy, also known as mitophagy, is an autophagosome-based mitochondrial degradation process that eliminates unwanted or damaged mitochondria after cell stress. Most studies dealing with mitophagy rely on the analysis by fluorescence microscopy of mitochondrial-autophagosome colocalization. However, given the fundamental role of mitophagy in the physiology and pathology of organisms, there is an urgent need for novel quantitative methods with which to study this process. Here, we describe a flow cytometry-based approach to determine mitophagy by using MitoTracker Deep Red, a widely used mitochondria-selective probe. Used in combination with selective inhibitors it may allow for the determination of mitophagy flux. Here, we test the validity of the use of this method in cell lines and in primary cell and tissue cultures.
Keywords: 3MA, 3-methyladenine; Atg, autophagy-related; CCCP, carbonyl cyanide m-chlorophenyl hydrazone; COX4I1, cytochrome c oxidase subunit IV isoform 1; Co, control; CsA, cyclosporin A; E, embryonic day; EBSS, Earle's balanced salt solution; FIS, fisetin; HCQ, hydroxychloroquine; KO, knockout; LC3, MAP1LC3/LC3; MEFs, mouse embryonic fibroblasts; MTDR, MitoTracker Deep Red; MTOR, mechanistic target of rapamycin; N+L, ammonium chloride + leupeptin; NAM, nicotinamide; P, postnatal day; PARK2, Parkin RBR E3 ubiquitin protein ligase; PHEN, 1,10-phenanthroline; PINK1, PTEN-induced putative kinase 1; Rapa, rapamycin; TIMM23, translocase of inner mitochondrial membrane 23 homolog (yeast); TOMM20, translocase of outer mitochondrial membrane 20 homolog (yeast); TOMM40, translocase of outer mitochondrial membrane 40 homolog (yeast); WM, wortmannin; WT, wild type; astrocyte primary culture; autophagic flux; autophagy; flow cytometry; mitochondria; mitophagic flux; mitophagy; neurodegeneration; retina; ΔΨ, mitochondrial membrane potential.