SUIT-006 Fluo mt D034

high-resolution terminology - matching measurements at high-resolution

SUIT-006 Fluo mt D034

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: orthogonal 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 NADH-linked substrates (type N-pathway to Q). Non-phosphorylating resting state (LEAK state); LEAK respiration L(n) in the absence of ADP, ATP, AMP (no adenylates).
2D	PMP	N	CI	1PM;2D OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
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 Uncoupler titration (avoiding inhibition by high uncoupler concentrations) to obtain electron transfer (ET) capacity E (noncoupled ET-state). Test for limitation of OXPHOS capacity P by the phosphorylation system (ANT, ATP synthase, phosphate transporter) relative to ET capacity E in mt-preparations: E-P control efficiency and E-L coupling efficiency. In living cells: E-R control efficiency and E-L coupling efficiency.
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).

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Coupling control

» Coupling control state

» ET capacity

» OXPHOS capacity

» LEAK respiration

Pathway control

» Electron transfer pathway

» Fatty acid oxidation pathway control state, F

» NADH electron transfer-pathway state, N

» Succinate pathway control state, S

» NS-pathway control state, NS

» Glycerophosphate pathway control state, Gp

» Complex IV single step, CIV

» Anaplerotic pathway control state

Main fuel substrates

» Glutamate, G

» Glycerophosphate, Gp

» Malate, M

» Octanoylcarnitine, Oct

» Pyruvate, P

» Succinate, S

Glossary

» List of SUIT states

» SUIT concept

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 assay without any sample to test the effect of the chemicals on the fluorescence signal. More information on our USB.

- 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 a expanded protocol to study mt-membrane potential not only with PM but also with glutamate and succinate.

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

MitoPedia concepts: SUIT protocol, SUIT A, Find 

MitoPedia methods: Fluorometry