SUIT-020 Fluo mt D033

high-resolution terminology - matching measurements at high-resolution

SUIT-020 Fluo mt D033

Description

Abbreviation: NS(PGM)

Reference: A: SUIT-020 short protocol for simultaneous determination of O₂ flux and mitochondrial membrane potential in mitochondrial preparations (isolated mitochondria and tissue homogenate).

SUIT number: D033_1PM;2D;3G;4S;5Rot;6Omy;7U;8Ama

O2k-Application: Fluo

SUIT-category: NS(PGM)

SUIT protocol pattern: orthogonal 1PM;2D;3G;4S;5Rot;6Omy;7U;8Ama

This is a protocol to investigate the N- and NS-pathway 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. Glutamate (G) and Succinate (S) are able to further increase the mt-membrane potential. Rotenone, which is an inhibitor of Complex I, depolarises the mt-membrane. 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. Antimycin A dissipates the mt-membrane potential.

Communicated by Huete-Ortega M, Komlodi T and Gnaiger E (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 NADH-linked substrates (type N-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
3G	PGMP	N	CI	1PM;2D;3G NADH-linked substrates (type N-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
4S	PGMSP	NS	CI&II	1PM;2D;3G;4S Respiratory stimulation by simultaneous action of type N substrates & succinate, with convergent electron flow in the NS-pathway for reconstitution of TCA cycle function. OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
5Rot	SP	S	CII	1PM;2D;3G;4S;5Rot Succinate, S ( type S-pathway to Q). Succinate pathway control state (S-pathway) after inhibiting CI with rotenone, which also inhibits the F-pathway.
6Omy	SL(Omy)			1PM;2D;3G;4S;5Rot;6Omy Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(Omy), after blocking the ATP synthase with oligomycin.
7U*	SE	S	CII	1PM;2D;3G;4S;5Rot;6Omy;7S Succinate, S ( type S-pathway to Q). 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. Noncoupled electron transfer state, ET state, with ET capacity E.
8Ama	ROX			1PM;2D;3G;4S;5Rot;6Omy;7S;8Ama 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.

+ You get information in a single protocol about the NS, S and N-pathway control state.

- 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-020 O2 mt D032.

- 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-004;SUIT-004 O2 pfi D010: provides an information about the NS(PM) pathway in the ET state withou the contribution of G.
• SUIT-008;SUIT-008 O2 pfi D014 and SUIT-008 O2 pce D025: provides information about the NS(PGM) pathway in the OXPHOS and ET-states, but without the addtion of Omy.
• SUIT-011: provides information about the NS(GM) pathway in the OXPHOS and ET-states, but without the addtion of Omy.
• SUIT-012: provides information about the N(PGM) pathway in the OXPHOS and ET-states wihtout the contribution of S-pathway and without the addtion of Omy.
• SUIT-014: a protocol similar to SUIT-020 O2 mt D035, but with the addition of P in the OXPHOS state before S. Omy is not added in the 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, Find 

MitoPedia methods: Fluorometry