Reference: A Coupling control protocol and Crabtree effect test -SUIT-003
SUIT number: D037_ce1;ce1Glc;(ce2Omy);ce3U;ce4Ama
SUIT-003 O2 ce D037 has been designed to study the regulation of the coupling control state and respiration by glucose (Crabtree effect) of intact cells on ROUTINE. To obtain more information about Crabtree effect, this protocol can be used in parallel with SUIT-003 O2 ce D038. SUIT-003 O2 ce D037 follows a modular approach and can be easily used in combination with the assessment of complex IV capacity and respirometric cell viability test. This protocol allows us to omit oligomycin, therefore the LEAK-respiration.If the aim is to analyse only Crabtree effetc, 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.
Communicated by Komlodi T, Garcia-Souza LF, Cardoso LH and Gnaiger E (last update 2019-04-03)
Steps and respiratory states
|Step||State||Pathway||Q-junction||Comment - Events (E) and Marks (M)|
- » Selected keywords in SUIT protocols
- Pathway control
Strengths and limitations
- The SUIT-003 O2 ce D037 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 D038. 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 uncoupler.
Compare SUIT protocols
- SUIT-003 O2 ce D038 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 and cell viability test.
- SUIT-003 O2 ce-pce D018 for assessment of coupling control, Crabtree effect, malate effect and cell viability test.
MitoPedia methods: Respirometry