SUIT-003 O2 ce D038

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SUIT-003 O2 ce D038

Description

Abbreviation: ceGlc

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

SUIT number: D038_ce1;ce2U;ce3Glc

O2k-Application: O2

MitoPedia: SUIT

SUIT-category: ceGlc

SUIT protocol pattern: complex cell coupling control protocol ce1;ce2U;ce3Glc

SUIT-003 O2 ce D038 has been designed to study the coupling control state and respiration regulation by glucose (Crabtree effect) of intact cells on ET-capacity control state. The different capacities are tested in the sequence OXPHOS and ET. SUIT-003 O2 ce D038 follows a modular approach and can be easily used in combination with the assessment of complex IV capacity and respirometric cell viability test.

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(Omy)			ce1;(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;(ce2Omy);ce3U Noncoupled electron transfer state, ET state, with ET capacity E.
ce3Glc	E			ce1;(ce2Omy);ce3U;ce3Glc Noncoupled electron transfer state, ET state, with ET capacity E. Glucose is added to test Crabtree effect.
ce3'U	E			ce1;(ce2Omy);ce3U;ce3Glc;ce3'U Noncoupled electron transfer state, ET state, with ET capacity E. Additional uncoupler titrations are performed to ensure reaching ET state after addition of glucose.
ce4Ama	ROX			ce1;(ce2Omy);ce3U;ce3Glc;ce3'U;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 D038 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).
• CIV activity can also be measured after the cell viability test module.

+ Reasonable duration of the experiment.

- Cell culture media with glucose (e.g. DMEM: 25 µM Glucose) could hinder the cell viability test.

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

Compare SUIT protocols

• SUIT-003 O2 ce-pce D013
• SUIT-003 O2 ce-pce D018
• SUIT-003 O2 ce D037

References

MitoPedia concepts: SUIT protocol, SUIT A, Find 

MitoPedia methods: Respirometry