Abbreviation: CCP-ce Crabtree_E
SUIT number: D038_ce1;(ce2Omy);ce3U;ce3Glc;ce3'U;ce4Ama
- 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)
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 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
- SUIT-003 O2 ce D037 can 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|
|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.
MitoPedia methods: Respirometry