SUIT-003 O2 ce D061

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SUIT-003 O2 ce D061

Description

Ce1;(ce2Omy);ce3U;ce4Rot;ce5DMSO;ce6DMSO;ce7Ama.png

Abbreviation: CCP-ce Snv,Mnanv - control

Reference: B Carrier control for the coupling-control protocol for living cells with cell permeable compounds MitoKit-CII/Succinate-nv and MitoKit-CII/Malonate-nv - SUIT-003 O2 ce D060

SUIT number: D061_ce1;(ce2Omy);ce3U;ce4Rot;ce5DMSO;ce6DMSO;ce7Ama

O2k-Application: O2

MitoPedia: SUIT - Coupling-control protocol (CCP) with the carrier of the plasma membrane-permeable succinate (MitoKit-CII/Succinate-nv) and plasma membrane-permeable malonate (MitoKit-CII/Malonate-nv) in living cells (ce): DMSO.
SUIT protocol pattern: ce1;(ce2Omy);ce3U;ce4DMSO;ce5DMSO;ce6Rot;ce7Ama

SUIT-003 O2 ce D061 protocol is a control for the SUIT-003 O2 ce D060, with the addition of the carrier DMSO instead of the plasma membrane-permeable succinate (MitoKit-CII/Succinate-nv) and plasma membrane-permeable malonate (MitoKit-CII/Malonate-nv). Moreover, the SUIT-003 O2 ce D061 protocol allows evaluating the coupling control of living cells. Respiratory capacities are tested in a sequence of coupling states: ROUTINE and Electron transfer pathway. 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. SUIT-003 O2 ce D061 protocol is a control for the SUIT-003 O2 ce D060, with the addition of the carrier DMSO instead of the plasma membrane-permeable succinate ([[[MitoKit-CII/Succinate-nv]]) and plasma membrane-permeable malonate (MitoKit-CII/Malonate-nv). Moreover, the SUIT-003 O2 ce D061 protocol allows evaluating the coupling control of living cells. Respiratory capacities are tested in a sequence of coupling states: ROUTINE and Electron transfer pathway. 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  Garcia-Souza LF, Cardoso LHD and Gnaiger E(last update 2019-07-02)
MitoPedia: SUIT

Steps and respiratory states

Ce1;(ce2Omy);ce3U;ce4Rot;ce5DMSO;ce6DMSO;ce7Ama.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(Omy) ce1;(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 cce1;(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.
ce5DMSO ROX ce1;(ce2Omy);ce3U;ce4Rot;ce5DMSO
  • 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.
ce6DMSO ROX ce1;(ce2Omy);ce3U;ce4Rot;ce5DMSO;ce6DMSO
  • 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.
ce7Ama ROX ce1;(ce2Omy);ce3U;ce4Rot;ce5DMSO;ce6DMSO;ce7Ama
  • 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.

Strengths and limitations

+ This protocol is a control for SUIT-003 O2 ce D060, with titrations of the carrier DMSO.
+ Reasonable duration of the experiment.
- To obtain LEAK respiration, the concentration 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