Cookies help us deliver our services. By using our services, you agree to our use of cookies. More information

SUIT-003 O2 ce-pce D018

From Bioblast
The printable version is no longer supported and may have rendering errors. Please update your browser bookmarks and please use the default browser print function instead.


high-resolution terminology - matching measurements at high-resolution


SUIT-003 O2 ce-pce D018

Description

Ce1;ce1P;ce2Omy;ce3U;ce4Glc;ce5Rot;ce6S;1Dig;1U;1c;2Ama;3AsTm;4Azd.png

Abbreviation: CCVP-Glc

Reference: C SUIT-003 Second version of the Coupling-control protocol and respirometric cell viability test (CCV protocol)

SUIT number: D018_ce1;ce1P;ce2Omy;ce3U;ce3Glc;ce4Rot;ce5S;1Dig;1c;2Ama;3AsTm;4Azd

O2k-Application: O2

SUIT protocol pattern: ce1;ce1P;ce2Omy;ce3U;ce3Glc;ce4Rot;ce5S;1Dig;1c;2Ama;3AsTm;4Azd

SUIT-003 O2 ce-pce D018 is designed to study coupling control and plasma membrane permeability of living cells. Respiratory capacities are tested in a sequence of coupling states: ROUTINE, LEAK respiration and Electron transfer pathway in the presence of glucose. To study LEAK respiration, the phosphorylation system is inhibited by oligomycin. The final concentration of oligomycin has to be carefully optimized for various cell types, to minimize the inhibitory effect on the electron transfer system which would lead to an underestimation of ET capacity. At Electron transfer pathway state, glucose is added to test the Crabtree effect on living cells. The viability index, VI, in the simple CCVP-Glc (SUIT-003 O2 ce-pce D018) may be underestimated if succinate uptake in the living cells proceeds through the dicarboxylate carrier of the plasma membrane. CCVP-Glc can be extended by the Complex IV module.

Communicated by Doerrier C, Garcia-Souza LF (last update 2019-06-06)


MitoPedia: SUIT

Steps and respiratory states

Ce1;ce1P;ce2Omy;ce3U;ce4Glc;ce5Rot;ce6S;1Dig;1U;1c;2Ama;3AsTm;4Azd.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.
ce1P ROUTINE ce1;ce1P; ROUTINE, R
  • ROUTINE respiration in the physiological coupling state R. Externally added permeable substrates could contribute to this respiratory state.
ce2Omy L(Omy) ce1;ce1P;ce2Omy
  • Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(Omy), after blocking the ATP synthase with oligomycin.
ce3U E ce1;ce1P;ce2Omy;ce3U
ce3Glc E ce1;ce1P;ce2Omy;ce3U;ce3Glc
ce4Rot ROX ce1;ce1P;ce2Omy;ce3U;ce3Glc;ce4Rot
  • 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.
ce5S ce1;ce1P;ce2Omy;ce3U;ce3Glc;ce4Rot;ce5S
1Dig SE ce1;ce1P;ce2Omy;ce3U;ce3Glc;ce4Rot;ce5S;1Dig
1Ama ROX ce1;ce1P;ce2Omy;ce3U;ce3Glc;ce4Rot;ce5S;1Dig;2Ama
  • Rox is the residual oxygen consumption in the ROX state, due to oxidative side reactions, estimated after addition of antimycin A (inhibitor of CIII). Rox is subtracted from oxygen flux as a baseline for all respiratory states, to obtain mitochondrial respiration (mt).


Step Respiratory state Pathway control ET-Complex Comment
## AsTm AsTmE CIV CIV
## Azd CHB


Questions.jpg


Click to expand or collaps

Strengths and limitations

  • The SUIT-003 O2 ce-pce D018 is a cell coupling-control protocol. The total cell count (Nce) is the sum of viable cells (Nvce) and dead cells (Ndce).
  • When MiR05 is used, the addition of pyruvate is recommended to provide an external fuel substrate.
  • CIV activity can also be measured after the cell viability test module.
+ With the extended module, S-ET capacity can be assessed after the performance of the coupling-control protocol, as well as outer mt-membrane integrity with the addition of cytochrome c.
- The addition of oligomycin has to be carefully tested to avoid inhibitory effects in the evaluation of the ET capacity.
- Careful washing is required after the experiment to avoid carry-over of inhibitors and uncoupler.


Compare SUIT protocols

References

MitoPedia concepts: SUIT protocol, SUIT C, Find 


MitoPedia methods: Respirometry