SUIT-003 O2 ce D062

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



MitoPedia

SUIT-003 O2 ce D062

Description

Ce1;ce1DMSO;(ce2Omy);ce3U;ce4Rot;ce5Ama.png

Abbreviation: CCP-ce Snv - control

Reference: A: Carrier control for the Coupling control protocol for living cells with plasma membrane-permeable succinate (MitoKit-CII/Succinate-nv) - SUIT-003 O2 ce D050

SUIT number: D062_ce1;ce1DMSO;(ce2Omy);ce3U;ce4Rot;ce5Ama

O2k-Application: O2

MitoPedia: SUIT - Coupling control protocol (CCP) with the carrier of the plasma membrane-permeable succinate (MitoKit-CII/Succinate-nv) in living cells (ce): DMSO.
SUIT protocol pattern: ce1;ce1DMSO;(ce2Omy);ce3U;ce4Rot;ce5Ama

SUIT-003 O2 ce D062 protocol is the control of the SUIT-003 O2 ce D050 adding the carrier DMSO in ROUTINE respiration instead of the plasma membrane-permeable succinate (MitoKit-CII/Succinate-nv). Moreover,the SUIT-003 O2 ce D062 protocol allows to evaluate the coupling control of living cells. Respiratory capacities are tested in a sequence of coupling states: ROUTINE and ET. Optionally, to study LEAK-respiration, the phosphorylation system might be 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.

Communicated by Doerrier C (last update 2019-06-26)
MitoPedia: SUIT

Steps and respiratory states

Ce1;ce1DMSO;(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.
ce1DMSO ROUTINE ce1
  • ROUTINE respiration in the physiological coupling state R. Externally added permeable substrates could contribute to this respiratory state.
(ce2Omy) L(Omy) ce1;ce1Snv;(ce2Omy)
  • Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(Omy), after blocking the ATP synthase with oligomycin.
  • The addition of oligomycin is optional depending on the experimental question to resolve.
ce3U E ce1;ce1Snv;(ce2Omy);ce3U
ce4Rot ROX ce1;ce1Snv;(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;ce1Snv;(ce2Omy);ce3U;ce4Rot;ce5Ama
  • 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.



Questions.jpg


Click to expand or collaps

Strengths and limitations

+ This is a cell coupling control protocol that provides information on ROUTINE and ET in the presence of (MitoKit-CII/Succinate-nv.
- This protocol is provided without oligomycin addition. To also include LEAK in the protocol, an event for the addition of oligomycin and a related mark can be added by editing the DLP file, creating a DLPU. However, 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

  • SUIT-003 O2 ce D050 the plasma membrane-permeable succinate (MitoKit-CII/Succinate-nv) is added in ROUTINE.


References

MitoPedia concepts: SUIT protocol, SUIT B, Find 


MitoPedia methods: Respirometry