Difference between revisions of "SUIT-006 Fluo mt D034"
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SUIT-006 Fluo mt D034 is a protocol to investigate the O<sub>2</sub> flux and [[mitochondrial membrane potential]] with fluorescent dyes. In this protocol, the [[NADH Electron transfer-pathway state]] can be analyzed in [[Mitochondrial preparations| 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. | SUIT-006 Fluo mt D034 is a protocol to investigate the O<sub>2</sub> flux and [[mitochondrial membrane potential]] with fluorescent dyes. In this protocol, the [[NADH Electron transfer-pathway state]] can be analyzed in [[Mitochondrial preparations| 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. | 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 | Since high concentrations of Omy can decrease the ET capacity induced by addition of uncoupler, the optimal concentration of Omy has to be determined. | ||
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Revision as of 15:57, 8 June 2020
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
- MitoPedia: SUIT
- 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.
Communicated by Komlodi T, Iglesias-Gonzalez J and 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
|
- Bioblast links: SUIT protocols - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>
- 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.
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,
Find
MitoPedia methods:
Fluorometry