Difference between revisions of "Krumschnabel 2015 Methods Mol Biol"
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|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. | |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 | |keywords=Amplex Red, Resorufin, High-resolution respirometry, Oxygraph, Respiration media, Substrate–uncoupler–inhibitor titration, Mouse brain mitochondria | ||
|mipnetlab=AT Innsbruck OROBOROS, | |mipnetlab=AT Innsbruck OROBOROS, AT Innsbruck Gnaiger E, SK Bratislava Sumbalova Z, AT Innsbruck MitoCom | ||
}} | }} | ||
{{Labeling | {{Labeling | ||
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|substratestates=CI, CII, CI&II, ROX | |substratestates=CI, CII, CI&II, ROX | ||
|instruments=Oxygraph-2k, O2k-Fluorometer | |instruments=Oxygraph-2k, O2k-Fluorometer | ||
|additional=O2k-Demo, O2k-MultiSensor | |||
}} | }} | ||
* '''O2k-Protocol''': »[[MiPNet19.20 AmplexRed H2O2-production]] | * '''O2k-Protocol''': »[[MiPNet19.20 AmplexRed H2O2-production]] | ||
Revision as of 16:27, 7 June 2015
 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 E (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, AT Innsbruck MitoCom
Labels: MiParea: Respiration, Instruments;methods
Stress:Oxidative stress;RONS Organism: Mouse Tissue;cell: Nervous system Preparation: Isolated mitochondria
Coupling state: LEAK, OXPHOS, ETS"ETS" is not in the list (LEAK, ROUTINE, OXPHOS, ET) of allowed values for the "Coupling states" property.
HRR: Oxygraph-2k, O2k-Fluorometer
O2k-Demo, O2k-MultiSensor
- O2k-Protocol: »MiPNet19.20 AmplexRed H2O2-production
