title: New method to assess mitophagy flux by flow cytometry.
abstract: 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.
authors.affiliation: a Departament of Cellular and Molecular Biology; Centro de Investigaciones Biológicas; CSIC ; Madrid , Spain.
authors.forename: Marta
authors.initials: M
authors.lastname: Mauro-Lizcano
authors.forename: Lorena
authors.initials: L
authors.lastname: Esteban-Martínez
authors.forename: Esther
authors.initials: E
authors.lastname: Seco
authors.forename: Ana
authors.initials: A
authors.lastname: Serrano-Puebla
authors.forename: Lucia
authors.initials: L
authors.lastname: Garcia-Ledo
authors.forename: Cláudia
authors.initials: C
authors.lastname: Figueiredo-Pereira
authors.forename: Helena L A
authors.initials: HL
authors.lastname: Vieira
authors.forename: Patricia
authors.initials: P
authors.lastname: Boya
mesh_terms: D000596:Amino Acids / Q000172:deficiency; D000818:Animals; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D004195:Disease Models, Animal; D015536:Down-Regulation / Q000187:drug effects; D004622:Embryo, Mammalian / Q000166:cytology; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005419:Flavonoids / Q000494:pharmacology; D044948:Flavonols; D005434:Flow Cytometry / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism; D008810:Mice, Inbred C57BL; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D063306:Mitophagy / Q000187:drug effects; D009536:Niacinamide / Q000494:pharmacology; D012160:Retina / Q000187:drug effects / Q000378:metabolism; D012174:Retinitis Pigmentosa / Q000473:pathology
chemical_list: D000596:Amino Acids; D005419:Flavonoids; D044948:Flavonols; D009536:Niacinamide; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; C017875:fisetin
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