Krumschnabel 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
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 Yeast
O2k-Protocol: »MiPNet20.14 AmplexRed H2O2-production
- 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
- Sobotka et al (2021) Facts and artefacts on the oxygen dependence of hydrogen peroxide production using Amplex UltraRed in yeast cells. MitoFit Preprints 2021.