SUIT-006 Fluo mt D034

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SUIT-006 Fluo mt D034

Description

1PM;2D;3Omy;4U;5Ama.png

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

SUIT-006 Fluo mt D034 O2.png SUIT-006 Fluo mt D034.png

MitoPedia: SUIT

Steps and respiratory states

1PM;2D;3Omy;4U;5Ama.png

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
  • Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(Omy), after blocking the ATP synthase with oligomycin.
  • The addition of oligomycin is optional depending on the experimental question to resolve. If it is known that Ln has the same values for LOmy for a kind of sample and substrates, this step may be skipped. If the carrier of oligomycin (EtOH) is used instead, it is possible to evaluate whether it affects OXPHOS. In these cases, LEAK state is not reached again.
4U PME N CI 1PM;2D;3Omy;4U
5Ama ROX 1PM;2D;3Omy;4U;5Ama
  • Rox is the residual oxygen consumption in the ROX state, due to oxidative side reactions, estimated after addition of antimycin A (inhibitor of CIII). Rox is subtracted from oxygen flux as a baseline for all respiratory states, to obtain mitochondrial respiration (mt).


Questions.jpg


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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 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

 YearReferenceOrganismTissue;cell
MiPNet20.13 Safranin mt-membranepotential2019-06-24
O2k-Protocols
O2k-FluoRespirometry: HRR and simultaneous determination of mt-membrane potential with safranin or TMRM.
MouseNervous system
Krumschnabel 2014 Methods Enzymol2014Krumschnabel 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.MouseNervous system




MitoPedia concepts: SUIT protocol, SUIT A, Find 


MitoPedia methods: Fluorometry