Description
Abbreviation: N(PM)
Reference: A: SUIT-006 short protocol for simultaneous determination of O2 flux and H2O2 in mitochondrial preparations (isolated mitochondria and tissue homogenate).
SUIT number: D048_1PM;2D;3Omy;4U;5Ama
O2k-Application: AmR
- SUIT-category: N(PM)
- SUIT protocol pattern: orthogonal 1PM;2D;3Omy;4U;5Ama
This is a protocol to investigate the N- control state in isolated mitochondria and tissue homogenate. Oligomycin (Omy) is used to induce a LEAK state of respiration via the inhibition of the ATP synthase. Since higher concentrations of Omy can decrease the ET state induced upon addition of uncoupler, the required concentration of Omy has to be determined. This is a short protocol to study simultaneously the O2 flux and H2O2 flux, in particular the dependence of H2O2 flux on the mt-membrane potantial. Addition of ADP depolarises the mt-membrane which leads to the decrease of the H2O2 flux. 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.
Communicated by Komlodi T (last update 2019-02-20)
Steps and respiratory states
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
0DTPA |
| |||
0SOD |
| |||
0HRP |
| |||
0AmR |
|
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
1PM | PML(n) | 1PM
| ||
2D | PMP | 1PM;2D
| ||
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
- Β» Electron transfer pathway
- Β» Fatty acid oxidation pathway control state, F
- Β» NADH electron transfer-pathway state, N
- Β» Succinate pathway control state, S
- Β» NS-pathway control state, NS
- Β» Glycerophosphate pathway control state, Gp
- Β» Complex IV single step, CIV
- Β» Anaplerotic pathway control state
- Pathway control
- Main fuel substrates
- Β» Glutamate, G
- Β» Glycerophosphate, Gp
- Β» Malate, M
- Β» Octanoylcarnitine, Oct
- Β» Pyruvate, P
- Β» Succinate, S
- Main fuel substrates
- Glossary
Strengths and limitations
- This protocol can be run under low oxygen concentration to test the oxygen dependency of mt-ROS production. For more technical details see: SUIT-018 AmR mt D031, SUIT-018 AmR mt D041.
- + Reasonable duration of the experiment.
- + 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.
- - CIV activity cannot be determined and Cytochrome c test cannot be performed together with the fluorescence dyes.
- - Omy concentration has to be determined. Higher concentrations of Omy may inhibit the ET state.
Compare SUIT protocols
References
Year | Reference | Organism | Tissue;cell | |
---|---|---|---|---|
MiPNet20.13 Safranin mt-membranepotential | 2019-06-24 | O2k-FluoRespirometry: HRR and simultaneous determination of mt-membrane potential with safranin or TMRM. | Mouse | Nervous system |
Krumschnabel 2014 Methods Enzymol | 2014 | Krumschnabel G, Eigentler A, Fasching M, Gnaiger E (2014) Use of safranin for the assessment of mitochondrial membrane potential by high-resolution respirometry and fluorometry. Methods Enzymol 542:163-81. https://doi.org/10.1016/B978-0-12-416618-9.00009-1 | Mouse | Nervous system |
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