SUIT-003 O2 ce-pce D018

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SUIT-003 O2 ce-pce D018

Description

Abbreviation: CCV protocol (old version)

Reference: C SUIT-003 Second version of the Coupling control protocol and respirometric cell viability test (CCV protocol)

SUIT number: D018_ce1;ce1P;ce2Omy;ce3U;ce3Glc;ce4Rot;ce5S;1Dig;1c;2Ama;3AsTm;4Azd

O2k-Application: O2

SUIT protocol pattern: ce1;ce1P;ce2Omy;ce3U;ce3Glc;ce4Rot;ce5S;1Dig;1c;2Ama;3AsTm;4Azd

Communicated by Doerrier C, Garcia-Souza LF and Iglesias-Gonzalez J (last update 2019-06-06)

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.
ce1P	ROUTINE			ce1;ce1P; ROUTINE, R ROUTINE respiration in the physiological coupling state R. Externally added permeable substrates could contribute to this respiratory state.
ce2Omy	L(Omy)			ce1;ce1P;ce2Omy Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(Omy), after blocking the ATP synthase with oligomycin.
ce3U	E			ce1;ce1P;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.
ce3Glc	E			ce1;ce1P;ce2Omy;ce3U;ce3Glc Noncoupled electron transfer state, ET state, with ET capacity E. Glucose is added to test Crabtree effect.
ce4Rot	ROX			ce1;ce1P;ce2Omy;ce3U;ce3Glc;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.
ce5S				ce1;ce1P;ce2Omy;ce3U;ce3Glc;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).
1Dig	SE			ce1;ce1P;ce2Omy;ce3U;ce3Glc;ce4Rot;ce5S;1Dig Multiple Digitonin titrations for complete plasma membrane permeabilization. Noncoupled electron transfer state, ET state, with ET capacity E.
1Ama	ROX			ce1;ce1P;ce2Omy;ce3U;ce3Glc;ce4Rot;ce5S;1Dig;2Ama 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).

Step	Respiratory state	Pathway control	ET-Complex	Comment
## AsTm	AsTmE	CIV	CIV
## Azd	CHB

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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 O2 ce-pce D018 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).
• When MiR05 is used, the addition of pyruvate is recommended to provide an external fuel substrate.
• CIV activity can also be measured after the cell viability test module.

+ 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.

- 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.

Compare SUIT protocols

• SUIT-003 O2 ce-pce D020 (A Advantageous alternative protocol)
• SUIT-003 O2 ce-pce D013

References

MitoPedia concepts: SUIT protocol, SUIT C, Find 

MitoPedia methods: Respirometry