SUIT-003 O2 ce-pce D018

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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

Communicated by Doerrier C, Garcia-Souza LF and Iglesias-Gonzalez J (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 entry into Q-junction Comment
## Tm TmE CIV CIV
## Azd ROX


Questions.jpg


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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