title: Use of potentiometric fluorophores in the measurement of mitochondrial reactive oxygen species.
abstract: Mitochondrial reactive oxygen species (ROS) are implicated in signal transduction, inflammation, neurodegenerative disorders, and normal aging. Net ROS release by isolated brain mitochondria derived from a mixture of neurons and glia is readily quantified using fluorescent dyes. Measuring intracellular ROS in intact neurons or glia and assigning the origin to mitochondria are far more difficult. In recent years, the proton-motive force crucial to mitochondrial function has been exploited to target a variety of compounds to the highly negative mitochondrial matrix using the lipophilic triphenylphosphonium cation (TPP(+)) as a "delivery" conjugate. Among these, MitoSOX Red, also called mito-hydroethidine or mito-dihydroethidium, is prevalently used for mitochondrial ROS estimation. Although the TPP(+) moiety of MitoSOX enables the manyfold accumulation of ROS-sensitive hydroethidine in the mitochondrial matrix, the membrane potential sensitivity conferred by TPP(+) creates a daunting set of challenges not often considered in the application of this dye. This chapter provides recommendations and cautionary notes on the use of potentiometric fluorescent indicators for the approximation of mitochondrial ROS in live neurons, with principles that can be extrapolated to nonneuronal cell types. It is concluded that mitochondrial membrane potential changes render accurate estimation of mitochondrial ROS using MitoSOX difficult to impossible. Consequently, knowledge of mitochondrial membrane potential is essential to the application of potentiometric fluorophores for the measurement of intramitochondrial ROS.
authors.affiliation: Department of Anesthesiology and Center for Shock, Trauma and Anesthesiology Research (STAR), University of Maryland School of Medicine, Baltimore, Maryland, USA; Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland, USA. Electronic address: bpolster@anes.umm.edu.
authors.forename: Brian M
authors.initials: BM
authors.lastname: Polster
authors.affiliation: Department of Clinical Sciences in Malmö, Unit of Molecular Metabolism, Lund University Diabetes Centre, CRC, Malmö, Sweden; Buck Institute for Research on Aging, Novato, California, USA.
authors.forename: David G
authors.initials: DG
authors.lastname: Nicholls
authors.affiliation: Department of Anesthesiology and Center for Shock, Trauma and Anesthesiology Research (STAR), University of Maryland School of Medicine, Baltimore, Maryland, USA.
authors.forename: Shealinna X
authors.initials: SX
authors.lastname: Ge
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland, USA; Center for Biomedical Engineering and Technology, University of Maryland School of Medicine, Baltimore, Maryland, USA.
authors.forename: Brian A
authors.initials: BA
authors.lastname: Roelofs
mesh_terms: D000818:Animals; D004996:Ethidium / Q000031:analogs & derivatives / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism; D053078:Membrane Potential, Mitochondrial; D008928:Mitochondria / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism; D010617:Phenanthridines / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D011199:Potentiometry / Q000379:methods*; D051381:Rats; D017382:Reactive Oxygen Species / Q000032:analysis* / Q000378:metabolism
chemical_list: C521281:5-(6'-triphenylphosphoniumhexyl)-5,6-dihydro-6-phenyl-3,8-phenanthridinediammine; D005456:Fluorescent Dyes; D010617:Phenanthridines; D017382:Reactive Oxygen Species; C067883:dihydroethidium; C058428:hydroethidine; D004996:Ethidium
keywords: Dihydroethidium; Hydroethidine; Membrane potential; MitoSOX; ROS; Respiration; Seahorse; Superoxide; Triphenylphosphonium; Uncoupling