Description
Abbreviation: CCP mt PM - Fluo
Reference: A: short protocol for simultaneous determination of O2 flux and mitochondrial membrane potential in permeabilized cells -SUIT-006
SUIT number: D097_ce1;1Dig;1PM;2D;3Omy;4U;5Ama
O2k-Application: Fluo
- SUIT-category: N(PM)
- SUIT protocol pattern: coupling-control protocol ce1;1Dig;1PM;2D;3Omy;4U;5Ama.png
SUIT-006 Fluo ce-pce D097 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 permeabilized cells (permeabilized by digitonin titration into the chamber). Addition of PM (pyruvate & malate) 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 depolarizes 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 Grings M, Cardoso Luiza HD (last update 2024-09-19)
Representative traces
Steps and respiratory states
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
ce1 | ROUTINE | ce1
| ||
1Dig | REN | ce1;1Dig
|
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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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 ce-pce D098: 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 ce-pce D099: this is a similar protocol to SUIT-020 Fluo ce-pce D098 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 |
---|---|---|
1Dig | Digitonin (Dig) |
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
MitoPedia concepts: SUIT protocol, SUIT A, Find
MitoPedia methods:
Fluorometry