Description
Abbreviation: CCP mt PM - Fluo
Reference: A: short protocol for simultaneous determination of O2 flux and mitochondrial membrane potential in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells) -SUIT-006
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
SUIT-006 Fluo mt D034 is a protocol to investigate the O2 flux and mitochondrial membrane potential with fluorescent dyes. In this protocol, the NADH Electron transfer-pathway state can be analyzed in mitochondrial preparations such as isolated mitochondria, tissue homogenates and permeabilized cells (already permeabilized when they are added to the chamber) in a wide variety of organisms and tissues. Addition of PM (pyruvate & malate) to the mitochondria leads to the hyperpolarisation of the mt-membrane, while ADP (D) decreases the mt-membrane potential. Addition of oligomycin (Omy) results in hyperpolarisation since the inhibition of the ATP synthase leads to an accumulation of protons in the intermembrane space. Uncoupler depolarises the mt-membrane in a concentration-dependent manner and antimycin A blocks the respiration and dissipates the mt-membrane potential. Since high concentrations of Omy can decrease the ET capacity induced by addition of uncoupler, the optimal concentration of Omy has to be determined. For mt-membrane potential analysis and calculation, visit this page: Mitochondrial membrane potential.
Communicated by Komlodi T, Gnaiger E (last update 2019-07-05)
Representative traces
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 fluorescence 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.
- Nigericin as a H+/K+ antiporter can be used to dissipate transmembrane pH gradient, which results in increased mt-membrane potential in the LEAK state.
- - Many fluorescence dyes (such as Safranin, TMRM, Rhodamine 123, etc) can inhibit components of the ETS, most commonly affecting NADH-linked respiration. Therefore, an experimental control should be done in the absence of the fluorescence dye to check for inhibitory effects. The following protocol is suggested: 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-021 Fluo mt D036: this is a similar protocol to SUIT-020 Fluo mt D033 for the samples which display a preference for GM instead of PM.
Chemicals and syringes
Step | Chemical(s) and link(s) | Comments |
---|---|---|
Saf | Safranin (Saf) |
or
Step | Chemical(s) and link(s) | Comments |
---|---|---|
TMRM | Tetramethylrhodamine methyl ester (TMRM) |
or
Step | Chemical(s) and link(s) | Comments |
---|---|---|
Rh123 | Rhodamine 123 |
Step | Chemical(s) and link(s) | Comments |
---|---|---|
1PM | Pyruvate (P) and Malate (M) | |
2D | ADP (D) | |
3Omy | Oligomycin (Omy) | |
4U | Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U) | Can be substituted for other uncoupler |
5Ama | Antimycin A (Ama) |
- Suggested stock concentrations are shown in the specific DL-Protocol.
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 |
MitoPedia concepts:
SUIT protocol,
SUIT A,
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MitoPedia methods:
Fluorometry