SUIT-003 O2 ce D037

high-resolution terminology - matching measurements at high-resolution

SUIT-003 O2 ce D037

Description

Abbreviation: CCP-ce-Crabtree(R)

Reference: A Coupling control protocol and Crabtree effect - SUIT-003

SUIT number: D037_ce1;ce1Glc;(ce2Omy);ce3U;ce4Ama

O2k-Application: O2

MitoPedia: SUIT

SUIT protocol pattern: ce1;ce1Glc;(ce2Omy);ce3U-

The CCP-ce-Crabtree (SUIT-003 O2 ce D037) is designed to study coupling control in living cells and the effect of glucose (or fructose; Crabtree effect) on ROUTINE respiration. This protocol allows us to omit oligomycin, therefore the LEAK-respiration. If the aim is to analyze only the Crabtree effect, it is recommended to skip the oligomycin step. If there is an interest in studying coupling control, oligomycin should be added to promote LEAK-respiration. To obtain more information about the Crabtree effect, it is ideal to use in parallel the protocol SUIT-003 O2 ce D038 (glucose addition in ET state). SUIT-003 O2 ce D037 can be extended by the cell viability test and the Complex IV module.

Communicated by Komlodi T, Garcia-Souza LF, Cardoso LH, Gnaiger E (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.
ce1Glc	ROUTINE			ce1;ce1Glc ROUTINE respiration in the physiological coupling state R. Externally added permeable substrates could contribute to this respiratory state. Glucose is added to test Crabtree effect.
(ce2Omy)	L(Omy)			ce1;ce1Glc;(ce2Omy) Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(Omy), after blocking the ATP synthase with oligomycin. If the aim is to analyse only Crabtree effect, it is recommended to skip this step. However, if there is also interest in studying coupling control, Omy can be added at this point to reach LEAK.
ce3U	E			ce1;ce1Glc;(ce2Omy);ce3U Noncoupled electron transfer state, ET state, with ET capacity E.
ce4Ama	ROX			ce1;ce1Glc;(ce2Omy);ce3U;ce4Ama 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 O2 ce D037 is a coupling control protocol for living cells. 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).
• CIV activity can also be measured after the cell viability test module.

+ Reasonable duration of the experiment.

+ To obtain more information about Crabtree effect, this protocol can be used in parallel with SUIT-003 O2 ce D038. In SUIT-003 O2 ce D037, the addition of glucose in the ROUTINE state can lead to an increase in ATP production by glycolysis, which can indirectly decrease respiration. In SUIT-003 O2 ce D038, with the addition of glucose in ET state, this effect is bypassed, and only the direct effect of glucose on the electron transfer system is tested.

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

Compare SUIT protocols

• SUIT-003 O2 ce D038 is recommended to be used in parallel to obtain more information on Crabtree effect.
• SUIT-003 O2 ce-pce D013 for assessment of coupling control, Crabtree effect, malate effect, and cell viability test.
• SUIT-003 O2 ce-pce D018 for assessment of coupling control, Crabtree effect, and cell viability test.

References

MitoPedia concepts: SUIT protocol, SUIT A, Find 

MitoPedia methods: Respirometry