Selective fluorescent imaging of superoxide
            <i>in vivo</i>
            using ethidium-based probes

Robinson, Kristine M.; Janes, Michael; Pehar, Mariana; Monette, Jeffrey S.; Ross, Meredith F.; Hagen, Tory M.; Murphy, Michael P.; Beckman, Joseph S.

doi:10.1073/pnas.0601945103

Cited by 672 publications

(624 citation statements)

References 37 publications

(54 reference statements)

Supporting

Mentioning

605

Contrasting

Unclassified

Order By: Relevance

“…While the possibility of formation of the hydroxylated cation during the reaction of Mito-HE with was suggested earlier [4], the authors could not distinguish between the two possible isomeric forms of the product (hydroxylation at the carbon atom C(2) or C (9)). The proposed structure of the product was 9-OH-Mito-E + .…”

Section: Discussionmentioning

confidence: 82%

“…1) is a selective probe for detecting superoxide [1,2]. HE reacts fairly rapidly with (k = 2 × 10 6 M −1 s −1 ) forming a unique red fluorescent marker product, 2-hydroxyethidium cation (2-OH-E + ) [3,4]. Results from these studies dispelled the long-held notion that superoxide reacts with HE to form ethidium (E + ) as a product.…”

Section: Introductionmentioning

confidence: 99%

“…Both 2-OH-E + and E + exhibit a "red fluorescence" that is enhanced by DNA [1,2]. Although subtle differences in the spectroscopic properties between E + and 2-OH-E + were used to selectively detect 2-OH-E + and E + in cells under well-defined conditions [4], the fluorescence techniques were largely deemed to be unsuitable for superoxide measurements in cells [2,5]. Using the HPLC-fluorescence method, it was previously shown that the increase in intracellular "red fluorescence" arising from HE could not be solely attributed to 2-OH-E + , as E + was also formed intracellularly from HE during oxidation [2,5].…”

Section: Introductionmentioning

confidence: 99%

“…1) that accumulated in mitochondria was synthesized [4]. This mitochondria-targeted probe was shown to react with superoxide forming a hydroxylated mito-ethidium cation, similar to that formed from the HE/superoxide reaction [4].…”

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Cytochrome c-mediated oxidation of hydroethidine and mito-hydroethidine in mitochondria: Identification of homo- and heterodimers

Zielonka

Srinivasan

Hardy

et al. 2008

Free Radical Biology and Medicine

134

View full text Add to dashboard Cite

Here we report that ferricytochrome c (cyt c 3+ ) induces oxidation of hydroethidine (HE) and mitochondria-targeted hydroethidine (Mito-HE or MitoSOX ™ Red) forming highly characteristic homo-and heterodimeric products. Using a HPLC-electrochemical (EC) method, several products were detected from cyt c 3+ -catalyzed oxidation of HE and Mito-HE and characterized by mass spectrometry and NMR techniques as follows: homodimers (HE-HE, E + -E + ; Mito-HE-Mito-HE, Mito-E + -Mito-E + ) and heterodimers (HE-E + and Mito-HE-Mito-E + ), as well as the monomeric ethidium (E + ) and mito-ethidium (Mito-E + ). Similar products were detected when HE and Mito-HE were incubated with mitochondria. In contrast, mitochondria depleted of cyt c 3+ were much less effective in oxidizing HE or Mito-HE to corresponding dimeric products. Unlike E + or Mito-E + , the dimeric analogs (E + -E + and Mito-E + -Mito-E + ) were not fluorescent. Superoxide ( ) or Fremy's salt react with Mito-HE to form a product, 2-hydroxy-mito-ethidium (2-OH-Mito-E + ) that was detected by HPLC. We conclude that HPLC-EC but not the confocal and fluorescence microscopy is a viable technique for measuring superoxide and cyt c 3+ -dependent oxidation products of HE and Mito-HE in cells. Superoxide detection using HE and Mito-HE could be severely compromised due to their propensity to undergo oxidation.

show abstract

Section: Discussionmentioning

confidence: 82%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Cytochrome c-mediated oxidation of hydroethidine and mito-hydroethidine in mitochondria: Identification of homo- and heterodimers

Zielonka

Srinivasan

Hardy

et al. 2008

Free Radical Biology and Medicine

134

View full text Add to dashboard Cite

show abstract

“…616 nm) and is often present at a much higher concentration (Zielonka and Kalyanaraman 2010). Discrimination between these two is still be possible, however, due to an extra excitation band between 350 and 400 nm for 2-OH-E + (Robinson et al 2006). However, as the ratio E + /2-OH-E + is often 10 or more, contribution of E + might still be significant ( Next to small molecule fluorescent probes, ROS can also be monitored using genetically encoded fluorescent protein based probes.…”

mentioning

confidence: 99%

Integrated High-Content Quantification of Intracellular ROS Levels and Mitochondrial Morphofunction

Sieprath

Corne

Willems

et al. 2016

Focus on Bio-Image Informatics

View full text Add to dashboard Cite

Oxidative stress arises from an imbalance between the production of reactive oxygen species (ROS) and their removal by cellular antioxidant systems. Especially under pathological conditions, mitochondria constitute a relevant source of cellular ROS. These organelles harbor the electron transport chain, bringing electrons in close vicinity to molecular oxygen. Although a full understanding is still lacking, intracellular ROS generation and mitochondrial function are also linked to changes in mitochondrial morphology. To study the intricate relationships between the different factors that govern cellular redox balance in living cells, we have developed a high-content microscopy-based strategy for simultaneous quantification of intracellular ROS levels and mitochondrial morphofunction. Here, we summarize the principles of intracellular ROS generation and removal, and we explain the major considerations for performing quantitative microscopy analyses of ROS and mitochondrial morphofunction in living cells. Next, we describe our workflow and finally, we illustrate that a multiparametric readout enables the unambiguous classification of chemically perturbed cells as well as laminopathy patient cells. Principles of intracellular ROS generation and removalReactive oxygen species (ROS) are small, short-lived derivatives of molecular oxygen (O 2 ) of radical and non-radical nature (Halliwell and Gutteridge 2007 • ), nitrate radical (NO 3 • ) and many other nitrogen derivatives. ROS were originally described as molecular constituents of the defense system of phagocytic cells, but it has become clear that besides their damaging properties, they also function as signaling molecules and mediate a variety of other cellular responses including cell proliferation, differentiation, gene expression and migration (Lambeth 2004;Bartz and Piantadosi 2010). Intracellular ROS metabolismROS can be generated at various sites in the cell (Fig. 1A). This can be either deliberately, e.g. by NADPH oxidases (NOX), or as a byproduct, e.g. during normal cellular respiration in mitochondria (Babior 1999;Turrens 2003;Murphy 2009). The NOX family of NADPH oxidases (NOX1, NOX2, NOX3, NOX4, NOX5, DUOX1 and DUOX2) are proteins that transport electrons (e -) from NADPH across biological membranes (plasma or endomembranes) (Bedard and Krause 2007;Dupre-Crochet et al. 2013). The activation mechanisms and tissue distribution of the isoforms differ but they all use O 2 as e --acceptor, producing O 2•-. Through ROS generation, they play a role in many cellular processes including host defense, regulation of gene expression and cell differentiation (Bedard and Krause 2007). Despite their sometimes significant contribution to the global ROS pools, NOX are not the predominant source of intracellular ROS. Mitochondria are considered the major culprit, in particular under pathological conditions. Mitochondrial ROS are generated as a byproduct of the oxidative phosphorylation (OXPHOS, cf. below). Irrespective of its source, ROS production generally sta...

show abstract

Assessing Mitochondrial Redox Status by Flow Cytometric Methods: Vascular Response to Fluid Shear Stress

Jen

et al. 2011

CP Cytometry

View full text Add to dashboard Cite

Mitochondria are an important source of superoxide production contributing to physiological and pathological responses, including vascular oxidative stress that is relevant to cardiovascular diseases. Vascular oxidative stress is intimately linked with pro‐inflammatory states and atherosclerosis. Oxidized low‐density lipoprotein (OxLDL) modulates intracellular redox status and induces apoptosis in endothelial cells. Hemodynamic, specifically, fluid shear stress imparts both biomechanical and metabolic effects on vasculature. Mitochondria are an important source of superoxide production contributing to vascular oxidative stress with relevance to cardiovascular diseases. We hereby present biophysical and biochemical approaches, including fluorescence‐activated cell sorting, to assess the dynamics of vascular redox status. Curr. Protoc. Cytom. 58:9.37.1‐9.37.14. © 2011 by John Wiley & Sons, Inc.

show abstract

Selective fluorescent imaging of superoxide in vivo using ethidium-based probes

Cited by 672 publications

References 37 publications

Cytochrome c-mediated oxidation of hydroethidine and mito-hydroethidine in mitochondria: Identification of homo- and heterodimers

Cytochrome c-mediated oxidation of hydroethidine and mito-hydroethidine in mitochondria: Identification of homo- and heterodimers

Integrated High-Content Quantification of Intracellular ROS Levels and Mitochondrial Morphofunction

Assessing Mitochondrial Redox Status by Flow Cytometric Methods: Vascular Response to Fluid Shear Stress

Contact Info

Product

Resources

About