Description
Abbreviation: N(PM)
Reference: A: short protocol for simultaneous determination of O2 flux and H2O2 in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells)- SUIT-006
SUIT number: D048_1PM;2D;3Omy;4U;5Ama
O2k-Application: AmR
- MitoPedia: SUIT
- SUIT-category: N(PM)
- SUIT protocol pattern: bidirectional linear coupling control protocol 1PM;2D;3Omy;4U;5Ama
SUIT-006 AmR mt D048 is a short protocol to study simultaneously the O2 flux and H2O2 flux (with the fluorescent dye Amplex UltraRed), in particular to study the dependence of H2O2 flux on the mt-membrane potential. In this protocol , the NADH Electron transfer-pathway state can be investigated in isolated mitochondria, tissue homogenate (except of liver homogenate) and permeabilized cells. After addition of the NADH-linked substrates, addition of ADP depolarises the mt-membrane which leads to the decrease of the H2O2 flux. Oligomycin (Omy) is used to induce a LEAK state of respiration via the inhibition of the ATP synthase. Omy hyperpolarises the mt-membrane resulting in increased H2O2 production, while the addition of the uncoupler decreases the H2O2 generation owing to the depolarisation of the mt-membrane. Since higher concentrations of Omy can decrease the ET state induced upon addition of uncoupler, the required concentration of Omy has to be determined.
Communicated by Komlodi T, Iglesias-Gonzalez J, Cardoso LH and Gnaiger E (last update 2019-04-02)
Steps and respiratory states
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
0DTPA |
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0SOD |
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0HRP |
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0AmR |
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Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
1PM | PML(n) | 1PM
| ||
2D | PMP | 1PM;2D
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3Omy | PML(Omy) | 1PM;2D;3Omy
| ||
4U | PME | 1PM;2D;3Omy;4U
| ||
5Ama | ROX | 1PM;2D;3Omy;4U;5Ama
|
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- Coupling control
- Pathway control
- Main fuel substrates
- » Glutamate, G
- » Glycerophosphate, Gp
- » Malate, M
- » Octanoylcarnitine, Oct
- » Pyruvate, P
- » Succinate, S
- Main fuel substrates
- Glossary
Strengths and limitations
- This protocol can be runned under low oxygen concentration to test the oxygen dependence of mt-ROS production. For more technical details see: SUIT-018 AmR mt D031, SUIT-018 AmR mt D041.
- + Reasonable duration of the experiment.
- - CIV activity cannot be determined and cytochrome c test cannot be performed together with the fluorescence dyes.
- + This protocol can be extended with the Complex IV module in the following protocols: SUIT-006 O2 mt D047.
- + Cytochrome c test can be performed in the following protocols:SUIT-006 O2 mt D047.
- - This protocol cannot be runned with liver homogenate.
- - Omy concentration has to be determined. Higher concentrations of Omy may inhibit the ET state.
Compare SUIT protocols
References
Year | Reference | Organism | Tissue;cell | |
---|---|---|---|---|
MiPNet24.10 H2O2 flux analysis | 2021-10-22 | Hydrogen peroxide flux analysis using Amplex UltraRed assay in MiR05-Kit with DatLab 7.4 | ||
Komlodi 2021 BEC AmR-O2 | 2021 | 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.org/10.26124/bec:2021-0004 | Saccharomyces cerevisiae | Other cell lines |
MiPNet20.14 AmplexRed H2O2-production | 2019-06-24 | O2k-FluoRespirometry: HRR and simultaneous determination of H2O2 production with Amplex UltraRed. | Mouse | Heart |
Komlodi 2018 Methods Mol Biol | 2018 | Komlodi T, Sobotka O, Krumschnabel G, Bezuidenhout N, Hiller E, Doerrier C, Gnaiger E (2018) Comparison of mitochondrial incubation media for measurement of respiration and hydrogen peroxide production. Methods Mol Biol 1782:137-55. | Human Mouse | Skeletal muscle HEK |
Krumschnabel 2015 Methods Mol Biol | 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. | Mouse | Nervous system |
Makrecka-Kuka 2015 Biomolecules | 2015 | 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. https://doi.org/10.3390/biom5031319 | Human Mouse | Heart Nervous system HEK |
MitoPedia concepts: SUIT protocol, SUIT A, Find
MitoPedia methods:
Fluorometry