SUIT-003 O2 ce D038

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SUIT-003 O2 ce D038

Description

Ce1;ce2(Omy);ce3U;ce3Glc;ce3'U;ce4Ama.png

Abbreviation: CCP-ce Crabtree_E

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

SUIT number: D038_ce1;(ce2Omy);ce3U;ce3Glc;ce3'U;ce4Ama

O2k-Application: O2

SUIT-category: ceGlc

SUIT-003 O2 ce D038 (CCP-ce Crabtree(E)) is designed to study coupling control in living cells and the effect of glucose (or fructose; Crabtree effect) on ET capacity. 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 D037 (glucose addition in ROUTINE). SUIT-003 O2 ce D038 can be extended by the cell viability test and the Complex IV module.

Communicated by Komlodi T , Garcia-Souza LF, Cardoso LHD and Gnaiger E (last update 2019-04-03)

Representative traces

D038 O2 trace.png

MitoPedia: SUIT

Steps and respiratory states

Ce1;ce2(Omy);ce3U;ce3Glc;ce3'U;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.
(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
ce3Glc E ce1;(ce2Omy);ce3U;ce3Glc
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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Strengths and limitations

  • The SUIT-003 O2 ce D038 is a cell coupling-control protocol. 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 D037. In SUIT-003 O2 ce D037, the addition of glucose in the ROUTINE state can lead to increase in ATP production by glycolysis, which can indirectly decrease respiration. In SUIT-003 O2 ce D038, with 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 inhibitors and uncoupler.

Compare SUIT protocols

Chemicals and syringes

Step Chemical(s) and link(s) Comments
(ce2Omy) Oligomycin (Omy) This step can be skipped
ce3U Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U) Can be substituted for other uncoupler
ce3Glc Glucose (Glc)
ce3'U Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U) Can be substituted for other uncoupler
ce4Ama Antimycin A (Ama)
Suggested stock concentrations are shown in the specific DL-Protocol.

References

MitoPedia concepts: SUIT protocol, SUIT A, Find 


MitoPedia methods: Respirometry