SUIT-003 O2 ce D062

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SUIT-003 O2 ce D062

Description

Abbreviation: Carrier for MitoKit-CII/Plasma membrane-permeable succinate (NV118)

Reference: A: - SUIT-003

SUIT number: D062_ce1;ce1DMSO;(ce2Omy);ce3U;ce4Rot;ce5Ama

O2k-Application: O2

MitoPedia: SUIT - Coupling control protocol (CCP) in living cells (ce) with the carrier of the plasma membrane-permeable succinate (MitoKit-CII/Succinate-nv): DMSO.

SUIT protocol pattern: ce1;ce1DMSO;(ce2Omy);ce3U;ce4Rot;ce5Ama

SUIT-003 O2 ce D062 protocol is the control of the SUIT-003 O2 ce D050 adding the carrier DMSO in ROUTINE respiration instead of the plasma membrane-permeable succinate (MitoKit-CII/Succinate-nv). Moreover,the SUIT-003 O2 ce D062 protocol allows to evaluate the coupling control of living cells. Respiratory capacities are tested in a sequence of coupling states: ROUTINE and ET. To study LEAK-respiration, the phosphorylation system is inhibited by oligomycin. The final concentration of oligomycin has to be carefully optimized for various cell types, to minimize the inhibitory effect on the electron transfer system which would lead to an underestimation of ET-capacity.

Communicated by Doerrier C (last update 2019-06-25)

Steps and respiratory states

Step	State	Comment - Events (E) and Marks (M)
ce1	ROUTINE	ce1 ROUTINE respiration in the physiological coupling state R. Externally added permeable substrates could contribute to this respiratory state.
ce1DMSO	ROUTINE	ce1 ROUTINE respiration in the physiological coupling state R. Externally added permeable substrates could contribute to this respiratory state.
(ce2Omy)	L(Omy)	ce1;ce1Snv;(ce2Omy) 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.
ce3U	E	ce1;ce1Snv;(ce2Omy);ce3U 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.
ce4Rot	ROX	ce1;ce1Snv;(ce2Omy);ce3U;ce4Rot Rox is the residual oxygen consumption in the ROX state, due to oxidative side reactions, estimated either after inhibition of CIII (e.g. antimycin A, myxothiazol), CIV (e.g. Cyanide) or in the absence of endogenous fuel-substrates. Rox is subtracted from oxygen flux as a baseline for all respiratory states, to obtain mitochondrial respiration.
ce5Ama	ROX	ce1;ce1Snv;(ce2Omy);ce3U;ce4Rot;ce5Ama Rox is the residual oxygen consumption in the ROX state, due to oxidative side reactions, estimated either after inhibition of CIII (e.g. antimycin A, myxothiazol), CIV (e.g. Cyanide) or in the absence of endogenous fuel-substrates. Rox is subtracted from oxygen flux as a baseline for all respiratory states, to obtain mitochondrial respiration.

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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

+ Reasonable duration of the experiment.

- To obtain LEAK respiration, the concentration of oligomycin has to be carefully tested to avoid inhibitory effects in the evaluation of the ET-capacity.

Compare SUIT protocols

SUIT-003 O2 ce D050 the plasma membrane-permeable succinate (MitoKit-CII/Succinate-nv) is added in ROUTINE.

References

MitoPedia concepts: SUIT protocol, SUIT B, Find

MitoPedia methods: Respirometry