SUIT-003 O2 ce D037

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SUIT-003 O2 ce D037

Description

Ce1;ce1Glc;ce2(Omy);ce3U;ce4Ama.png

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 SUIT-003 O2 ce D037 (CCP-ce Crabtree(R)) 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, Antunes D, Garcia-Souza LF, Cardoso LH, Gnaiger E (last update 2019-07-30)

Representative traces

D037 O2 traces.png

MitoPedia: SUIT

Steps and respiratory states

Ce1;ce1Glc;ce2(Omy);ce3U;ce4Ama.png

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
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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Strengths and limitations

  • The SUIT-003 O2 ce D037 is a coupling control protocol for living cells. The total cell count (Nce) is the sum of viable cells (Nvce) and dead cells (Ndce).
  • 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.

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References

MitoPedia concepts: SUIT protocol, SUIT A, Find 


MitoPedia methods: Respirometry