SUIT-003 O2 ce D038

From Bioblast
Jump to: navigation, search
Bioblasts - Richard Altmann and MiPArt by Odra Noel
MitoPedia         Terms and abbreviations         Concepts and methods         Oroboros O2k         MiP and biochemistry         Preprints and history



MitoPedia

SUIT-003 O2 ce D038

Description

Ce1;ce2(Omy);ce3U;ce3Glc;ce3'U;ce4Ama.png

Abbreviation: ceGlc

Reference: A Coupling control protocol and Crabtree effect test -SUIT-003

SUIT number: D038_ce1;(ce2Omy);ce3U;ce3Glc;ce3'U;ce4Ama

O2k-Application: O2

MitoPedia: SUIT
SUIT-category: ceGlc
SUIT protocol pattern: complex cell coupling control protocol ce1;(ce2Omy);ce3U;ce3Glc;ce3'U-

SUIT-003 O2 ce D038 has been designed to study the regulation of coupling control state and respiration by glucose (Crabtree effect) of intact cells on ET-capacity. To obtain more information about Crabtree effect, this protocol can be used in parallel with SUIT-003 O2 ce D037. SUIT-003 O2 ce D038 follows a modular approach and can be easily used in combination with the assessment of complex IV capacity and respirometric cell viability test. If the aim is to analyse only 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.


Communicated by Komlodi T , Garcia-Souza LF, Cardoso LH and Gnaiger E (last update 2019-04-03)
MitoPedia: SUIT

Steps and respiratory states

Ce1;ce2(Omy);ce3U;ce3Glc;ce3'U;ce4Ama.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.
(ce2Omy) L(Omy) ce1;(ce2Omy)
  • Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(Omy), after blocking the ATP synthase with oligomycin.
  • If the aim is to analyse only Crabtree effect, it is recommended to skip this step. However, if there is also interest in studying coupling control, Omy can be added at this point to reach LEAK.
ce3U E ce1;(ce2Omy);ce3U
ce3Glc E ce1;(ce2Omy);ce3U;ce3Glc
ce3'U E ce1;(ce2Omy);ce3U;ce3Glc;ce3'U
  • Noncoupled electron transfer state, ET-state, with ET-capacity, E.
  • Additional uncoupler titrations are performed to ensure reaching ET state after addition of glucose.
ce4Ama ROX ce1;(ce2Omy);ce3U;ce3Glc;ce3'U;ce4Ama
  • Rox is the residual oxygen consumption in the ROX state, due to oxidative side reactions, estimated either after inhibition of CIII (e.g. antimycin A, myxothiazol), CIV (e.g. Cyanide) or in the absence of endogenous fuel-substrates. Rox is subtracted from oxygen flux as a baseline for all respiratory states, to obtain mitochondrial respiration.


Questions.jpg


Click to expand or collaps

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

References

MitoPedia concepts: SUIT protocol, SUIT A, Find 


MitoPedia methods: Respirometry