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: CCVP

Reference: A Coupling control protocol and respirometric cell viability test; ce: living 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 protocol pattern: ce1;ce1P;ce2Omy;ce3U;ce4Rot;ce5S;1Dig;1c;2Ama;3AsTm;4Azd

SUIT-003 O2 ce-pce D020 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 and ET. 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. The viability index, VI, in the simple CCVP-ce (SUIT-003 O2 ce-pce D020) may be underestimated, if succinate uptake in the living cells proceeds throught the dicarboxylate carrier of the plasma membrane. CCVP-ce can be extended by the Complex IV module.

Communicated by Iglesias-Gonzalez J, Krumschnabel G, Doerrier C, Cardoso LH, Gnaiger E (last update 2019-06-06)
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
  • 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
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


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Strengths and limitations

  • The SUIT-003 O2 ce-pce D020 is a coupling control protocol for living cells. 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 instead of rotenone to reach Rox.
+ 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.

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References

  • Stadlmann S, Renner K, Pollheimer J, Moser PL, Zeimet AG, Offner FA, Gnaiger E (2006) Preserved coupling of oxidative phosphorylation but decreased mitochondrial respiratory capacity in IL-1ß treated human peritoneal mesothelial cells. Cell Biochem Biophys 44:179-86. - »Bioblast link«


MitoPedia concepts: SUIT protocol, SUIT A, Find 


MitoPedia methods: Respirometry