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SUIT-003 O2 ce D012

From Bioblast


high-resolution terminology - matching measurements at high-resolution


SUIT-003 O2 ce D012

Description

Ce1;ce1P;ce2Omy;ce3U;ce4Rot;ce5Ama.png

Abbreviation: CCP-ce(P)

Reference: B - SUIT-003

SUIT number: D012_ce1;ce1P;ce2Omy;ce3U;ce4Rot;ce5Ama

O2k-Application: O2


SUIT-003 O2 ce D012 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 pyruvate is recommended to support the cell respiration if the incubation media is not cell culture media. 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 known side effects over the ET-capacity. Rotenone and Antimycin A provide the ROX state and its sequential addition allows the extension of the protocol with the cell viability test module (See CCV SUIT-003 O2 ce-pce D020).

Communicated by Garcia-Souza LF, Gnaiger E (last update 2019-06-24)
MitoPedia: SUIT

Steps and respiratory states

Ce1;ce1P;ce2Omy;ce3U;ce4Rot;ce5Ama.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 L ce1;ce1P;ce2Omy

Non-phosphorylating resting state (LEAK state); LEAK respiration L(n) in the absence of ADP, ATP, AMP (no adenylates).


ce3U E ce1;ce1P;ce2Omy;ce3U

Uncoupler titration (avoiding inhibition by high uncoupler concentrations) to obtain electron transfer (ET) capacity E (noncoupled ET-state). Test for limitation of OXPHOS capacity P by the phosphorylation system (ANT, ATP synthase, phosphate transporter) relative to ET capacity E in mt-preparations: E-P control efficiency and E-L coupling efficiency. In living cells: E-R control efficiency and E-L coupling efficiency. Noncoupled electron transfer state, ET state, with ET capacity E.

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.
ce5Ama ROX ce1;ce1P;ce2Omy;ce3U;ce4Rot;ce5Ama
  • 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).

Strengths and limitations

  • The SUIT-003 O2 ce D012 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).
  • When the cell viability test module is not incorporated to the experiment, complex III inhibitors can be used to reach Rox instead of rotenone.
  • 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.
+ 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).
- 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 B, Find 


MitoPedia methods: Respirometry