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. Complex III inhibitor Antimycin (Ama) blocks the respiration. Addition of PM (Pyruvate & Malate) to the mitochondria leads to the hyperpolarisation of the mt-membrane, while ADP (D) decreases the mt-membrane pontial. Addition of Omy results in hyperpolarisation owing to the fact that the inhibition of the ATP synthase leads to accumulation of protons in the intermembrane space. Uncoupler depolarises the mt-membrane in a concentration-dependent manner and antimycin A dissipates the mt-membrane potential.
Communicated by Komlodi T (last update 2019-02-20)
Steps and respiratory states
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
1PM | PML(n) | N | CI | 1PM
|
2D | PMP | N | CI | 1PM;2D
|
3Omy | PML(Omy) | N | CI | 1PM;2D;3Omy
|
4U | PME | N | CI | 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 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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