SUIT-020 Fluo mt D033

high-resolution terminology - matching measurements at high-resolution

SUIT-020 Fluo mt D033

Description

Abbreviation: NS(PGM)

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

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-

SUIT-020 Fluo mt D033 is a protocol to assess the additivity between N-pathway and S- pathway in the Q-junction as well as investigate the N- and NS-pathway control state in mitochondrial preparations. It can serve as a diagnostic tool for the activity of glutamate dehydrogenase and its linked pathway. 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 assessed by the Omy titration test. Addition of PM (Pyruvate & Malate) to the mitochondria leads to the hyperpolarisation of the mt-membrane, while ADP (D) decreases the mt-membrane potential. 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, therefore leading to dissipation of the mt-membrane potential. Complex III inhibitor Antimycin A (Ama) blocks the respiration and dissipates the mt-membrane potential. For mt-membrane potential analysis and calculation, visit this page: Mitochondrial membrane potential.

Communicated by Huete-Ortega M, Antunes D, Komlodi T and Gnaiger E (last update 2019-07-25) 

Representative traces

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 without any sample to test the effect of the chemicals on the fluorescence signal. More information on our USB.
• NS-OXPHOS capacity provides a physiologically relevant estimate of maximum mitochondrial respiratory capacity.
• Comparison of GM- with PM-capacity yields important information on N-pathway respiratory control upstream of CI.
• A succinate concentration of >10 mM may be required for saturating SE capacity.
• 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.

+ You obtain 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.

- To test the effect of the fluorescence dye on the respiration, in SUIT-020 O2 mt D032 and SUIT-021 O2 mt D035 the dye can be added after cytochrome c.

- CIV activity cannot be determined and Cytochrome c test cannot be performed together with the fluorescence dyes.

- Oligomycin concentration has to be determined. Higher concentrations of Omy may inhibit the ET state.

- Careful washing is required after the experiment to avoid carry-over of the inhibitors and uncoupler.

Compare SUIT protocols

• SUIT-004;SUIT-004 O2 pfi D010: provides information about the NS(PM) pathway in the ET state without the contribution of G.
• SUIT-008;SUIT-008 O2 pfi D014 and SUIT-008 O2 ce-pce D025: provides information about the NS(PGM) pathway in the OXPHOS and ET-states, but without the addition of Omy.
• SUIT-011: provides information about the NS(GM) pathway in the OXPHOS and ET-states, but without the addition of Omy.
• SUIT-012: provides information about the N(PGM) pathway in the OXPHOS and ET-states without the contribution of S-pathway and without the addition of Omy.
• SUIT-014: a protocol similar to SUIT-021 O2 mt D035, but with the addition of P in the OXPHOS state before S. Omy is not added in the protocol.

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)
3G	Glutamate (G)
4S	Succinate (S)
5Rot	Rotenone (Rot)
6Omy	Oligomycin (Omy)
7U	Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U)	Can be substituted for other uncoupler
8Ama	Antimycin A (Ama)

Suggested stock concentrations are shown in the specific DL-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