SUIT-003 O2 ce D028

high-resolution terminology - matching measurements at high-resolution

SUIT-003 O2 ce D028

Description

Abbreviation: ceS

Reference: B SUIT-003 coupling control protocol with succinate for permeability test

SUIT number: D028_ce1;ce2Omy;ce3U;ce4Rot;ce5S;ce6Ama

O2k-Application: O2

SUIT-category: ceS

SUIT protocol pattern: linear cell coupling control protocol ce1;ce2Omy;ce3U;ce4Rot;ce5S;ce6Ama

References

Steps and respiratory states

Step	State	Pathway	Q-junction	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.
ce2Omy	L			ce1;ce2Omy Non-phosphorylating resting state (LEAK state); LEAK respiration L(n) in the absence of ADP, ATP, AMP (no adenylates).
ce3U	E			ce1;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;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.

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
ce5S				ce1;ce2Omy;ce3U;ce4Rot;ce5S Succinate pathway control state (S-pathway) after inhibiting CI with rotenone, which also inhibits the F-pathway. Succinate, S ( type S-pathway to Q).
ce6Ama	ROX			ce1;ce2Omy;ce3U;ce4Rot;ce5S;ce6Ama 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). 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

• The SUIT-003 Ce1;ce2Omy;ce3U;ce4Rot;ce5S;ce6Ama is a cell coupling control protocol. The total cell count (N_ce) is the sum of viable cells (N_vce) and dead cells (N_dce).
• This protocol can be extended with the cell viability test module (See CCV SUIT-003 O2 ce-pce D020).
• For determination of respirometric viability index, it is assumed that both viable and dead cells contain functional mitochondria.

+ With the extended module, S-ET capacity can be assessed after the performance of the coupling control protocol, as well as outer mt-membrane integrity with the addition of cytochrome c.

+ CIV activity can also be measured after the cell permeability test module.

+ Flexibility of experiment duration: from 1 hour (cell coupling control protocol) to 2 hours (permeability test module and CIV module).

- Cells with a cell membrane succinate transporter are not suitable for the viability or permeability test.

- The addition of oligomycin has to be carefully tested to avoid inhibitory effects in the evaluation of the ET-capacity.

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

ceR,L,E format

In the diagrams of coupling control protocols for intact cells (CCP), the steps may be replaced by the respiratory states.

• ce4Rot,ce5S;ce6Ama

ce4Rot: In many intact cells ROX measured after titration of rotenone (ce4Rot) is not or only slightly further inhibited by subsequent titration of antimycin A.

ce5S: Succinate is not permeable through the plasma membrane of many intact cells (Steinlechner-Maran 1997 Transplantation). If succinate is accessible to rotenone-inhibited uncoupled mitochondria in dead cells (dce), respiration will be stimulated.

ce6Ama: Inhibition of CIII by antimycin A inhibits succinate-stimulated respiration (Stadlmann_2006_Cell Biochem Biophys).

MitoPedia concepts: SUIT protocol, SUIT B, Find 

MitoPedia methods: Respirometry