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Use of potentiometric fluorophores in the measurement of mitochondrial reactive oxygen species.

Polster, Brian M; Nicholls, David LU ; Ge, Shealinna X and Roelofs, Brian A (2014) In Methods in Enzymology 547. p.225-250
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... (More)
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. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Methods in Enzymology
volume
547
pages
225 - 250
publisher
Academic Press
external identifiers
  • PMID:25416361
  • Scopus:84925384701
ISSN
0076-6879
DOI
10.1016/B978-0-12-801415-8.00013-8
language
English
LU publication?
yes
id
36195278-8c5e-4ee2-958c-f0b57557bb21 (old id 4816305)
alternative location
http://www.ncbi.nlm.nih.gov/pubmed/25416361?dopt=Abstract
date added to LUP
2014-12-03 15:03:13
date last changed
2016-11-24 15:00:54
@misc{36195278-8c5e-4ee2-958c-f0b57557bb21,
  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.},
  author       = {Polster, Brian M and Nicholls, David and Ge, Shealinna X and Roelofs, Brian A},
  issn         = {0076-6879},
  language     = {eng},
  pages        = {225--250},
  publisher    = {ARRAY(0xabdf670)},
  series       = {Methods in Enzymology},
  title        = {Use of potentiometric fluorophores in the measurement of mitochondrial reactive oxygen species.},
  url          = {http://dx.doi.org/10.1016/B978-0-12-801415-8.00013-8},
  volume       = {547},
  year         = {2014},
}