SUIT-023

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SUIT-023

Description

Ce1;ce2SHAM;ce3KCN.png

Abbreviation: ce

Reference: A: Determination of the respiration due to the cytochrome c oxidase pathway Bioblast pdf »Versions

SUIT-category: ce
SUIT protocol pattern: linear ce1;ce2SHAM;ce3KCN

The SUIT-023 is designed to provide an estimation of the mitochondrial respiration in cells due to the cytochrome c oxidase (CIV) pathway, which is non-resistant to cyanide-and antimycin A inhibition. This protocol must be complemented with the study in parallel of the mitochondrial respiration due to the alternative oxidase pathway (see SUIT-022).

Communicated by Huete-Ortega M and Gnaiger E (last update 2019-04-09)

Specific SUIT protocols

SUIT-023 O2 ce D053

Ce1;ce2SHAM;ce3KCN.png Ce1;ce2SHAM;ce3KCN.v2.png D053 O2 traces.png

MitoPedia: SUIT

Steps and respiratory states

Ce1;ce2SHAM;ce3KCN.png Ce1;ce2SHAM;ce3KCN.v2.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.
ce2SHAM ROUTINE OR ROX ce1;ce2SHAM
  • Salicylhydroxamic acid (SHAM) is an inhibitor of the alternative oxidase (AOX). When AOX is blocked by SHAM, electrons are forced through the cytochrome c oxidase (CIV) pathway.
  • After inhibition with SHAM, the ROUTINE state is normally achieved, defined as the respiration in the physiological coupling state where the mitochondrial respiration is through the cytochrome c oxidase (CIV) pathway (CIV). Occassionally, the cytochrome c oxidase pathway may not be active and then we have the ROX state, where Rox is the residual oxygen consumption due to oxidative side reactions estimated either after inhibition of CIII or CIV 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.
ce3KCN ROX ce1;ce2SHAM;ce3KCN


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Strengths and limitations

+ This protocol can be used with many type of cells, although optimal concentrations of inhibitors have to be optimised.
+ Short duration of experiment
- Estimation of the linear coupling control is not done
- The use of cyanide requires a careful and in deep cleaning of the chambers after use and careful handling of the chemical to guarantee health safety.

Compare SUIT protocols


References

MitoPedia concepts: MiP concept, SUIT protocol, Recommended 


MitoPedia methods: Respirometry