Difference between revisions of "Krumschnabel 2015 Methods Mol Biol"
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|title=Krumschnabel G, Fontana-Ayoub M, Sumbalova Z, Heidler J, Gauper K, Fasching M, Gnaiger EĀ (2015) Simultaneous high-resolution measurement of mitochondrial respiration and hydrogen peroxide production. Methods Mol Biol 1264:245-61. | |title=Krumschnabel G, Fontana-Ayoub M, Sumbalova Z, Heidler J, Gauper K, Fasching M, Gnaiger EĀ (2015) Simultaneous high-resolution measurement of mitochondrial respiration and hydrogen peroxide production. Methods Mol Biol 1264:245-61. | ||
|info=[http://www.ncbi.nlm.nih.gov/pubmed/25631019 PMID: 25631019] | |info=[http://www.ncbi.nlm.nih.gov/pubmed/25631019 PMID: 25631019] | ||
|authors=Krumschnabel G, Fontana-Ayoub M,, Sumbalova Z, Heidler J, Gauper K, Fasching M, Gnaiger | |authors=Krumschnabel G, Fontana-Ayoub M,, Sumbalova Z, Heidler J, Gauper K, Fasching M, Gnaiger Erich | ||
|year=2015 | |year=2015 | ||
|journal=Methods Mol Biol | |journal=Methods Mol Biol | ||
|abstract=[[Image:O2k-Protocols.jpg|right|80px|link=O2k-Protocols|O2k-Protocols contents]] | |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>ā¢-</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. | Mitochondrial respiration is associated with the formation of reactive oxygen species, primarily in the form of superoxide (O2<sup>ā¢-</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. | ||
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|keywords=Amplex Red, Resorufin, High-resolution respirometry, Oxygraph, Respiration media, Substrateāuncouplerāinhibitor titration, Mouse brain mitochondria | |keywords=Amplex Red, Resorufin, High-resolution respirometry, Oxygraph, Respiration media, Substrateāuncouplerāinhibitor titration, Mouse brain mitochondria | ||
|mipnetlab=AT Innsbruck OROBOROS, AT Innsbruck Gnaiger E, SK Bratislava Sumbalova Z | |mipnetlab=AT Innsbruck OROBOROS, AT Innsbruck Gnaiger E, SK Bratislava Sumbalova Z | ||
| Line 19: | Line 20: | ||
|pathways=N, S, NS, ROX | |pathways=N, S, NS, ROX | ||
|instruments=Oxygraph-2k, O2k-Fluorometer, O2k-Protocol | |instruments=Oxygraph-2k, O2k-Fluorometer, O2k-Protocol | ||
|additional=O2k-Demo, O2k-MultiSensor | |additional=O2k-Demo, O2k-MultiSensor, 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, AmR, H2O2 | ||
}} | }} | ||
[[Image:O2k-Protocols.jpg|40px|left|link=O2k-Protocols|O2k-Protocols]] '''O2k-Protocol''': Ā»[[MiPNet20.14 AmplexRed H2O2-production]] | [[Image:O2k-Protocols.jpg|40px|left|link=O2k-Protocols|O2k-Protocols]] '''O2k-Protocol''': Ā»[[MiPNet20.14 AmplexRed H2O2-production]] | ||
| Line 29: | Line 30: | ||
::::# Bouitbir J, Singh F, Charles AL, Schlagowski AI, Bonifacio A,Ā Echaniz-Laguna A, Geny B, KrƤhenbĆ¼hl S, Zoll J (2016) Statins triggerĀ mitochondrial ROS-induced apoptosis in glycolytic skeletal muscle. Antioxid Redox Signal 24:84-98. - [[Bouitbir 2016 Antioxid Redox Signal |Ā»Bioblast linkĀ«]] | ::::# Bouitbir J, Singh F, Charles AL, Schlagowski AI, Bonifacio A,Ā Echaniz-Laguna A, Geny B, KrƤhenbĆ¼hl S, Zoll J (2016) Statins triggerĀ mitochondrial ROS-induced apoptosis in glycolytic skeletal muscle. Antioxid Redox Signal 24:84-98. - [[Bouitbir 2016 Antioxid Redox Signal |Ā»Bioblast linkĀ«]] | ||
::::* and many other citations: The most frequently cited chapter of the book (2019-04-02). - [http://www.bookmetrix.com/detail_full/book/b1d5f986-6bcf-4afc-8eab-381b39639771#citations ''More details''] | ::::* and many other citations: The most frequently cited chapter of the book (2019-04-02). - [http://www.bookmetrix.com/detail_full/book/b1d5f986-6bcf-4afc-8eab-381b39639771#citations ''More details''] | ||
::{{Template:Cited by | ::{{Template:Cited by Komlodi 2021 MitoFit AmR-O2}} | ||
::{{Template:Cited by Komlodi 2021 MitoFit Tissue normoxia}} | ::{{Template:Cited by Komlodi 2021 MitoFit Tissue normoxia}} | ||
== O2k-Publications == | == O2k-Publications == | ||
* [[O2k-Publications: Instruments;methods]] | * [[O2k-Publications: Instruments;methods]] | ||
* [[O2k-Publications: O2k-Fluorometry]] | * [[O2k-Publications: O2k-Fluorometry]] | ||
== Cited by == | |||
::* 42 articles in PubMed (2021-12-27) https://pubmed.ncbi.nlm.nih.gov/25631019/ | |||
Latest revision as of 12:31, 25 October 2023
| 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. |
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 (O2ā¢-) 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.
ā¢ Keywords: Amplex Red, Resorufin, High-resolution respirometry, Oxygraph, Respiration media, Substrateāuncouplerāinhibitor titration, Mouse brain mitochondria
ā¢ O2k-Network Lab: AT Innsbruck OROBOROS, AT Innsbruck Gnaiger E, SK Bratislava Sumbalova Z
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, 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, AmR, H2O2
O2k-Protocol: Ā»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. - Ā»Bioblast linkĀ«
- Bouitbir J, Singh F, Charles AL, Schlagowski AI, Bonifacio A, Echaniz-Laguna A, Geny B, KrƤhenbĆ¼hl S, Zoll J (2016) Statins trigger mitochondrial ROS-induced apoptosis in glycolytic skeletal muscle. Antioxid Redox Signal 24:84-98. - Ā»Bioblast linkĀ«
- and many other citations: The most frequently cited chapter of the book (2019-04-02). - More details
-
- KomlĆ³di 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)
- 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
Cited by
- 42 articles in PubMed (2021-12-27) https://pubmed.ncbi.nlm.nih.gov/25631019/
