Abbreviation: CCP-ce Crabtree_R
SUIT number: D037_ce1;ce1Glc;(ce2Omy);ce3U;ce4Ama
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)
Steps and respiratory states
|Step||State||Pathway||Q-junction||Comment - Events (E) and Marks (M)|
- Bioblast links: SUIT protocols - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>
- Pathway control
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.
Compare SUIT protocols
- SUIT-003 O2 ce D038 is recommended to be used in parallel to obtain more information on 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|
|(ce2Omy)||Oligomycin (Omy)||This step can be skipped|
|ce3U||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.
MitoPedia methods: Respirometry