Difference between revisions of "SUIT-006 Fluo mt D034"
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{{MitoPedia | {{MitoPedia | ||
|abbr=N(PM) | |abbr=N(PM) | ||
|description=[[File:1PM;2D;3Omy;4U;5Ama.png|300px]] | |description=[[File:1PM;2D;3Omy;4U;5Ama.png|300px]][[File:SUIT-006 Fluo mt D034 O2.png|500px]][[File:SUIT-006 Fluo mt D034.png|500px]] | ||
|info='''A''': '''[[SUIT-006]]''' short protocol for simultaneous determination of O<sub>2</sub> flux and mitochondrial membrane potential in mitochondrial preparations (isolated mitochondria and tissue homogenate). | |info='''A''': '''[[SUIT-006]]''' short protocol for simultaneous determination of O<sub>2</sub> flux and mitochondrial membrane potential in mitochondrial preparations (isolated mitochondria and tissue homogenate). | ||
|application=Fluo | |application=Fluo |
Revision as of 09:53, 28 February 2019
Description
Abbreviation: N(PM)
Reference: A: SUIT-006 short protocol for simultaneous determination of O2 flux and mitochondrial membrane potential in mitochondrial preparations (isolated mitochondria and tissue homogenate).
SUIT number: D034_1PM;2D;3Omy;4U;5Ama
O2k-Application: Fluo
- SUIT-category: N(PM)
- SUIT protocol pattern: bidirectional linear coupling control protocol 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
- Before performing this protocol, a calibration with the fluoresence dye needs to be done. More information on our USB: Instrumental Protocols/Fluo calibration.
- It is recommended to run a chemical background without any sample to test the effect of the chemicals on the fluorescence signal.
- - Many fluorescence dyes (such as Safranin, TMRM etc) can inhibit components of the ET system, most commonly affecting NADH-linked respiration. Therefore, a control run of the protocol should be done in the absence of the fluorescence dye. The following protocol can be used: 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
- SUIT-020 Fluo mt D033: this is an expanded protocol to study mt-membrane potential not only with PM but also with glutamate and succinate to support NS(PGM)-pathway.
- SUIT-020 Fluo mt D036: this is a similar protocol to SUIT-020 Fluo mt D033 for the samples which display preference for GM instead of PM.
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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MitoPedia methods:
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