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: AmR

SUIT-category: NS(PGM)

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

Communicated by Komlodi T  (last update 2019-01-28)

DLP applications

• SUIT-020 Fluo mt D033 simultaneous determination of O₂ flux and mt-membrane potential using any fluorescence dye (Safranin, TMRM etc) on isolated mitochondria and tissue homogenate.
• SUIT-020 Fluo mt D036 simultaneous determination of O₂ flux and mt-membrane potential using any fluorescence dye (Safranin, TMRM etc) on isolated mitochondria and tissue homogenate.

• SUIT-020 O2 mt D032 determination of O₂ flux on isolated mitochondria and tissue homogenate as a control for SUIT-020 Safr mt D033.
• SUIT-020 O2 mt D035 determination of O₂ flux on isolated mitochondria and tissue homogenate as a control for SUIT-020 Safr mt D036.

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

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

+ Information in one protocol about the NS, S and N-pathway control state.

- The fluorescence dye (such as Safranin, TMRM etc) can inhibit most porbably the NADH-linked respiration, therefore, the protocol has to be run without using the fluorescence dye. The following protocols can be used: SUIT-020 O2 mt D032, SUIT-020 O2 mt D035.

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

- Omy concentration has to be determined. Higher concentration of Omy than required can inhibit the ET state.

Compare SUIT protocols

• SUIT-004;SUIT-004 O2 pfi D010: it 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: it provides information about the NS(PGM) pathway in the OXPHOS and ET-states, but without the addtion of Omy.
• SUIT-011:it provides information about the NS(GM) pathway in the OXPHOS and ET-states, but without the addtion of Omy.
• SUIT-012:it 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: it is similar protocol 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.

MitoPedia concepts: SUIT protocol, SUIT A, Find 

MitoPedia methods: Fluorometry