SUIT-003 O2 ce D012

From Bioblast
Jump to: navigation, search
Bioblasts - Richard Altmann and MiPArt by Odra Noel
MitoPedia         Terms and abbreviations         Concepts and methods         Oroboros O2k         MiP and biochemistry         Preprints and history



MitoPedia

SUIT-003 O2 ce D012

Description

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

Abbreviation: ceN(P)

Reference: B - SUIT-003 coupling control protocol (CCP) with pyruvate

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

O2k-Application: O2

SUIT-category: ceN(P)
SUIT protocol pattern: complex cell coupling control protocol ce1;ce1P;ce2Omy;ce3U;ce*

References

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 (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;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


MitoPedia concepts: SUIT protocol, SUIT B, Find 


MitoPedia methods: Respirometry