Abbreviation: CCP-Crabtree with glycolysis inhibition
SUIT number: D067_ce1;ce1Glc;ce2Omy;ce3DG;ce4Ama
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.
Steps and respiratory states
|Step||State||Pathway||Q-junction||Comment - Events (E) and Marks (M)|
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- Pathway control
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
- SUIT-003 O2 ce D038 and SUIT-003 O2 ce D037 for assessment for assessment of coupling control and 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.
Chemicals and syringes
|Step||Chemical(s) and link(s)||Comments|
|ce4Ama||Antimycin A (Ama)|
- Suggested stock concentrations are shown in the specific DL-Protocol.
MitoPedia methods: Respirometry