SUIT-003 O2 ce D028

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SUIT-003 O2 ce D028

Description

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

Abbreviation: ceS

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-category: ceS
SUIT protocol pattern: linear cell coupling control protocol ce1;ce2Omy;ce3U;ce4Rot;ce5S;ce6Ama

References

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

Uncoupler titration (avoiding inhibition by high uncoupler concentrations) to obtain electron transfer (ET) capacity (noncoupled ET-state), as a test for limitation of OXPHOS-capacity by the phosphorylation system (ANT, ATP synthase, phosphate transporter) relative to ET-capacity, E. Noncoupled electron transfer state, ET-state, with ET-capacity, E.

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

Succinate pathway control state (S-pathway) after inhibiting CI with rotenone, which also inhibits the F-pathway if fatty acids are present. Succinate, S ( type S-pathway to Q).


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).


MitoPedia concepts: SUIT protocol, SUIT B, Find 


MitoPedia methods: Respirometry