SUIT-003 O2 ce-pce D020

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SUIT-003 O2 ce-pce D020

Description

Ce1;ce1P;ce2Omy;ce3U;ce4Rot;ce5S;1Dig;1c;2Ama;3AsTm;4Azd.png

Abbreviation: ceP,S

Reference: A Coupling control protocol and respirometric cell viability test (CCV protocol); ce: non-permeabilized cells, pce: permeabilized cells - SUIT-003

SUIT number: D020_ce1;ce1P;ce2Omy;ce3U;ce4Rot;ce5S;1Dig;1c;2Ama;3AsTm;4Azd

O2k-Application: O2

MitoPedia: SUIT
SUIT-category: ceP,S
SUIT protocol pattern: complex cell coupling control protocol ce1;ce1P;ce2Omy;ce3U;ce4Rot;ce5S;1Dig;1c;2Ama;3AsTm;4Azd

SUIT-003 O2 ce-pce D020 has been designed to study the coupling control state and membrane permeability of intact cells. The different capacities are tested in the sequence OXPHOS, LEAK and ET. Also, in order to study the LEAK-respiration, we need to inhibit the phosphorylation system which in our protocol is achieved using oligomycin. However, the final concentration of oligomycin has to be carefully adjusted in preliminary experiments for different samples and, the inhibitory effect on the electron transfer system, has to be studied to avoid an underestimation of the ET-capacity. SUIT-003 O2 ce-pce D020 follows a modular approach and can be easily used in combination with the assessment of complex IV capacity.


Communicated by Iglesias-Gonzalez J, Krumschnabel G, Doerrier C and Cardoso LH (last update 2019-03-28)
MitoPedia: SUIT

Steps and respiratory states

Ce1;ce1P;ce2Omy;ce3U;ce4Rot;ce5S;1Dig;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. Pyruvate, ROUTINE, R
  • ROUTINE respiration in the physiological coupling state R. Externally added permeable substrates could contribute to this respiratory state.
ce2Omy LOmy 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
ce4Rot ROX ce1;ce1P;ce2Omy;ce3U;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.
Step State Pathway Q-junction Comment - Events (E) and Marks (M)
ce5S ce1;ce1P;ce2Omy;ce3U;ce4Rot;ce5S
1Dig* SE S CII ce1;ce1P;ce2Omy;ce3U;ce4Rot;ce5S;1Dig
1c SE S CII ce1;ce1P;ce2Omy;ce3U;ce4Rot;ce5S;1Dig;1U;1c
  • Addition of cytochrome c yields a test for integrity of the mtOM. Stimulation by added cytochrome c would indicate an injury of the mtOM and limitation of respiration in the preceding state without added c due to loss of cytochrome c. Typically, cytochrome c is added immediately after the earliest ADP-activation step (P, OXPHOS-capacity with saturating [ADP]).
  • Noncoupled electron transfer state, ET-state, with ET-capacity, E.
2Ama ROX ce1;ce1P;ce2Omy;ce3U;ce4Rot;ce5S;1Dig;1U;1c;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 D020 is a cell coupling control protocol. The total cell count (Nce) is the sum of viable cells (Nvce) and dead cells (Ndce).
  • For determination of respirometric viability index, it is assumed that both viable and dead cells contain functional mitochondria.
  • When the cell viability test module is not incorporated into the experiment, complex III inhibitors can be used to reach Rox instead of rotenone.
+ Respirometric cell viability test correlates with other cell viability quantification methods.
+ 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.
+ CIV activity can also be measured after the cell viability test module.
+ Flexibility of experiment duration: from 1 hour (cell coupling control protocol) to 2 and a half hours (cell coupling control, cell viability test module and CIV module).
- Cells with a cell membrane succinate transporter are not suitable for the viability test.
- Pathological profiles could hinder the cell viability test.
- Careful washing is required after the experiment to avoid carry-over of inhibitors and uncoupler.
- The concentration of oligomycin has to be carefully tested to avoid inhibitory effects in the evaluation of the ET-capacity.

Compare SUIT protocols

References

MitoPedia concepts: SUIT protocol, SUIT A, Find 


MitoPedia methods: Respirometry