title: Oxidative phosphorylation measurement in cell lines and tissues.
abstract: Mitochondrial oxidative phosphorylation (OXPHOS) dysfunction is implicated in a growing spectrum of diseases, from neurodegeneration to cancer. Where tissues or transformed cells are available, respirometry and enzymology allow a sophisticated analysis of OXPHOS with modest-cost equipment. The isolation of organelle fractions is also invaluable for determining association of proteins of interest. Here we revisit and consolidate methods to measure whole cell mitochondrial ATP synthesis, respiration, isolation of mitochondria from cultured cells and tissues, and OXPHOS enzymology. We also explain common pitfalls, guide optimisation of the methods for new users, and provide full laboratory protocols in Supplementary materials.
authors.affiliation: Centre for Eye Research Australia, 32 Gisborne St, East Melbourne, VIC 3002, Australia; Department of Ophthalmology, University of Melbourne, 32 Gisborne St, East Melbourne, VIC 3002, Australia; Royal Victorian Eye and Ear Hospital, 32 Gisborne St, East Melbourne, VIC 3002, Australia.
authors.forename: Nicole J
authors.initials: NJ
authors.lastname: Van Bergen
authors.affiliation: Centre for Eye Research Australia, 32 Gisborne St, East Melbourne, VIC 3002, Australia; Department of Ophthalmology, University of Melbourne, 32 Gisborne St, East Melbourne, VIC 3002, Australia; Royal Victorian Eye and Ear Hospital, 32 Gisborne St, East Melbourne, VIC 3002, Australia.
authors.forename: Rachel E
authors.initials: RE
authors.lastname: Blake
authors.affiliation: Centre for Eye Research Australia, 32 Gisborne St, East Melbourne, VIC 3002, Australia; Department of Ophthalmology, University of Melbourne, 32 Gisborne St, East Melbourne, VIC 3002, Australia; Royal Victorian Eye and Ear Hospital, 32 Gisborne St, East Melbourne, VIC 3002, Australia.
authors.forename: Jonathan G
authors.initials: JG
authors.lastname: Crowston
authors.affiliation: Centre for Eye Research Australia, 32 Gisborne St, East Melbourne, VIC 3002, Australia; Department of Ophthalmology, University of Melbourne, 32 Gisborne St, East Melbourne, VIC 3002, Australia; Royal Victorian Eye and Ear Hospital, 32 Gisborne St, East Melbourne, VIC 3002, Australia; Department of Medicine, St. Vincent's Hospital, 41 Victoria Parade, Fitzroy, VIC 3065, Australia. Electronic address: i.trounce@unimelb.edu.au.
authors.forename: Ian A
authors.initials: IA
authors.lastname: Trounce
mesh_terms: D002460:Cell Line; D003584:Cytological Techniques / Q000379:methods*; D006801:Humans; D008928:Mitochondria / Q000378:metabolism*; D010085:Oxidative Phosphorylation
keywords: ATP synthesis; Enzymology; Mitochondria; Oxidative phosphorylation; Protocol; Respiration