SUIT-003 O2 ce D067

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SUIT-003 O2 ce D067

Description

SUIT-003 O2 ce D067 diagram.png

Abbreviation: CCP-Crabtree with glycolysis inhibition

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

SUIT number: D067_ce1;ce1Glc;ce2Omy;ce3DG;ce4Ama

O2k-Application: O2


The SUIT-003 O2 ce D067 is designed to study the effect of glucose (or fructose; Crabtree effect) on ROUTINE respiration and glycolysis inhibition on LEAK-respiration simultaneously measured with proton flux in living cells. If the aim is to analyze only the Crabtree effect and the effect of 2-Deoxyglucose on proton flux and respiration via glycolysis inhibition, Oligomycin can be omitted. 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) and SUIT-003 O2 ce D037 (glucose addition in ROUTINE). The latter can be extended by the cell viability test and the Complex IV module.

Communicated by Komlodi T, [ Gnaiger E (last update 2020-02-20)

Representative traces

D067 O2 trace.pngD067 pH trace.png

MitoPedia: SUIT

Steps and respiratory states

SUIT-003 O2 ce D067 diagram.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
ce2Omy LOmy ce1;ce1Glc;ce2Omy
  • Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(Omy), after blocking the ATP synthase with oligomycin.
ce3DG L ce1;ce1Glc;ce2Omy;ce3DG
  • 2-Deoxyglucose inhibits glycolysis which is reflected in decreased respiration, acidification and proton flux.


Questions.jpg


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Strengths and limitations

  • The SUIT-003 O2 ce D067 is protocol for living cells to study Crabtree effect and glycolysis inhibition by measuring respiration and proton flux simultaneously.
  • 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 and SUIT-003 O2 ce D037. 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 Omy.

Compare SUIT protocols

Chemicals and syringes

Step Chemical(s) and link(s) Comments
ce1Glc Glucose (Glc)
ce2Omy Oligomycin (Omy)
ce3DG 2-Deoxyglucose (2-DG)
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