SUIT-003 O2 ce D028

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SUIT-003 O2 ce D028

Description

Ce1;ce2Omy;ce3U;ce4Rot;ce5S;ce6Ama.png

Abbreviation: CCP-ce S permeability test

Reference: B SUIT-003 coupling control protocol with succinate for permeability test

SUIT number: D028_ce1;ce2Omy;ce3U;ce4Rot;ce5S;ce6Ama

O2k-Application: O2


SUIT-003 O2 ce D028 is a coupling control protocol for living cells. Different coupling control states are assessed (ROUTINE - L(Omy) - E) at ce-substrate state. The use of oligomycin provides important information about the ETS and TCA activity but the optimal concentration has to be carefully determined experimentally to avoid the well-known side effects over the ET-capacity. After the addition of Rotenone to reach ROX state we add succinate, which does not permeate the plasma membrane, to test if the plasma membrane of the cells is altered (permeability test). If we do not obtain further stimulation of the respiration we could conclude that the living cells are not permeable and remain intact.

Communicated by Doerrier C, Iglesias-Gonzalez J and Gnaiger E (last update 2019-06-26)
MitoPedia: SUIT

Steps and respiratory states

Ce1;ce2Omy;ce3U;ce4Rot;ce5S;ce6Ama.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 ce1;ce2Omy
  • Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(n), in the absence of ADP, ATP, AMP (no adenylates).
ce3U E ce1;ce2Omy;ce3U
ce4Rot ROX ce1;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;ce2Omy;ce3U;ce4Rot;ce5S
ce6Ama ROX ce1;ce2Omy;ce3U;ce4Rot;ce5S;ce6Ama
  • 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).
  • 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.


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Strengths and limitations

  • The SUIT-003 Ce1;ce2Omy;ce3U;ce4Rot;ce5S;ce6Ama 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).
  • For determination of respirometric viability index, it is assumed that both viable and dead cells contain functional mitochondria.
+ 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 permeability test module.
+ Flexibility of experiment duration: from 1 hour (cell coupling control protocol) to 2 hours (permeability test module and CIV module).
- Cells with a cell membrane succinate transporter are not suitable for the viability or permeability test.
- 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.

ceR,L,E format

R;L;E format.jpg In the diagrams of coupling control protocols for intact cells (CCP), the steps may be replaced by the respiratory states.

  • ce4Rot,ce5S;ce6Ama
ce4Rot: In many intact cells ROX measured after titration of rotenone (ce4Rot) is not or only slightly further inhibited by subsequent titration of antimycin A.
ce5S: Succinate is not permeable through the plasma membrane of many intact cells (Steinlechner-Maran 1997 Transplantation). If succinate is accessible to rotenone-inhibited uncoupled mitochondria in dead cells (dce), respiration will be stimulated.
ce6Ama: Inhibition of CIII by antimycin A inhibits succinate-stimulated respiration (Stadlmann_2006_Cell Biochem Biophys).

References

MitoPedia concepts: SUIT protocol, SUIT B, Find 


MitoPedia methods: Respirometry