Difference between revisions of "Krumschnabel 2015 Methods Mol Biol"

Revision as of 11:13, 27 November 2021

Krumschnabel G, Fontana-Ayoub M, Sumbalova Z, Heidler J, Gauper K, Fasching M, Gnaiger E (2015) Simultaneous high-resolution measurement of mitochondrial respiration and hydrogen peroxide production. Methods Mol Biol 1264:245-61.

» PMID: 25631019

Krumschnabel G, Fontana-Ayoub M, Sumbalova Z, Heidler J, Gauper K, Fasching M, Gnaiger Erich (2015) Methods Mol Biol

Abstract:

Mitochondrial respiration is associated with the formation of reactive oxygen species, primarily in the form of superoxide (O2^•-) and particularly hydrogen peroxide (H₂O₂). Since H₂O₂ plays important roles in physiology and pathology, measurement of hydrogen peroxide has received considerable attention over many years. Here we describe how the well-established Amplex Red assay can be used to detect H₂O₂ production in combination with the simultaneous assessment of mitochondrial bioenergetics by high-resolution respirometry. Fundamental instrumental and methodological parameters were optimized for analysis of the effects of various substrate, uncoupler and inhibitor titrations (SUIT) on respiration versus H₂O₂ production. The sensitivity of the H₂O₂ assay was strongly influenced by compounds contained in different mitochondrial respiration media, which also exerted significant effects on chemical background fluorescence changes. Near-linearity of the fluorescence signal was restricted to narrow ranges of accumulating resorufin concentrations independent of the nature of mitochondrial respiration media. Finally, we show an application example using isolated mouse brain mitochondria as an experimental model for the simultaneous measurement of mitochondrial respiration and H₂O₂ production in SUIT protocols.

• Keywords: Amplex Red, Resorufin, High-resolution respirometry, Oxygraph, Respiration media, Substrate–uncoupler–inhibitor titration, Mouse brain mitochondria

• O2k-Network Lab: AT Innsbruck OROBOROS, AT Innsbruck Gnaiger E, SK Bratislava Sumbalova Z

O2k-Protocol: »MiPNet20.14 AmplexRed H2O2-production

O2k-Publication

Cited by

Makrecka-Kuka M, Krumschnabel G, Gnaiger E (2015) High-resolution respirometry for simultaneous measurement of oxygen and hydrogen peroxide fluxes in permeabilized cells, tissue homogenate and isolated mitochondria. Biomolecules 5:1319-38. - »Bioblast link«
Bouitbir J, Singh F, Charles AL, Schlagowski AI, Bonifacio A, Echaniz-Laguna A, Geny B, Krähenbühl S, Zoll J (2016) Statins trigger mitochondrial ROS-induced apoptosis in glycolytic skeletal muscle. Antioxid Redox Signal 24:84-98. - »Bioblast link«

and many other citations: The most frequently cited chapter of the book (2019-04-02). - More details

Komlódi T, Sobotka O, Gnaiger E (2021) Facts and artefacts on the oxygen dependence of hydrogen peroxide production using Amplex UltraRed. Bioenerg Commun 2021.4. https://doi:10.26124/BEC:2021-0004

Komlodi et al (2022) Hydrogen peroxide production, mitochondrial membrane potential and the coenzyme Q redox state measured at tissue normoxia and experimental hyperoxia in heart mitochondria. MitoFit Preprints 2021 (in prep)

O2k-Publications

Labels: MiParea: Respiration, Instruments;methods

Stress:Oxidative stress;RONS Organism: Mouse Tissue;cell: Nervous system Preparation: Isolated mitochondria

Coupling state: LEAK, OXPHOS, ET Pathway: N, S, NS, ROX HRR: Oxygraph-2k, O2k-Fluorometer, O2k-Protocol

O2k-Demo, O2k-MultiSensor, Amplex Ultra Red, SUIT-009, SUIT-009 AmR mt D021, SUIT-009 AmR pce D019, SUIT-013, SUIT-013 AmR ce D023, SUIT-018, SUIT-018 AmR mt D031, SUIT-006 AmR mt D048, SUIT-003 AmR ce D017, SUIT-003 AmR ce D058, MitoFit 2021 AmR, MitoFit 2021 AmR-O2, MitoFit 2021 Tissue normoxia

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 |abstract=[[Image:O2k-Protocols.jpg|right|80px|link=O2k-Protocols|O2k-Protocols contents]]
 Mitochondrial respiration is associated with the formation of reactive oxygen species, primarily in the form of superoxide (O2<sup>•-</sup>) and particularly hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>). Since H<sub>2</sub>O<sub>2</sub> plays important roles in physiology and pathology, measurement of hydrogen peroxide has received considerable attention over many years. Here we describe how the well-established Amplex Red assay can be used to detect H<sub>2</sub>O<sub>2</sub> production in combination with the simultaneous assessment of mitochondrial bioenergetics by high-resolution respirometry. Fundamental instrumental and methodological parameters were optimized for analysis of the effects of various substrate, uncoupler and inhibitor titrations (SUIT) on respiration versus H<sub>2</sub>O<sub>2</sub> production. The sensitivity of the H<sub>2</sub>O<sub>2</sub> assay was strongly influenced by compounds contained in different mitochondrial respiration media, which also exerted significant effects on chemical background fluorescence changes. Near-linearity of the fluorescence signal was restricted to narrow ranges of accumulating resorufin concentrations independent of the nature of mitochondrial respiration media. Finally, we show an application example using isolated mouse brain mitochondria as an experimental model for the simultaneous measurement of mitochondrial respiration and H<sub>2</sub>O<sub>2</sub> production in SUIT protocols.
+<br><br>
 |keywords=Amplex Red, Resorufin, High-resolution respirometry, Oxygraph, Respiration media, Substrate–uncoupler–inhibitor titration, Mouse brain mitochondria
 |mipnetlab=AT Innsbruck OROBOROS, AT Innsbruck Gnaiger E, SK Bratislava Sumbalova Z