SUIT-023

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SUIT-023

Description

Ce1;ce2SHAM;ce3KCN.png

Abbreviation: AOX-ce SHAM+CN

Reference: A: Determination of respiration through the CIII-CIV pathway

SUIT protocol pattern: ce1;ce2SHAM;ce3KCN

The SUIT-023 provides an estimation of mitochondrial respiration in living cells through the CIII- cytochrome c oxidase (CIV) pathway, which is inhibited by antimycin A and cyanide. This protocol must be complemented with the study in parallel of mitochondrial respiration through the Q-alternative oxidase pathway (Q-AOX; see SUIT-022).

Communicated by Huete-Ortega M, Gnaiger E (last update 2019-06-05)

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 types of cells, although optimal concentrations of inhibitors have to be tested.
+ Short duration of experiment
- No estimation of coupling control
- The use of cyanide requires careful cleaning of the chambers after use and careful handling of the chemical to guarantee health safety.


Compare SUIT protocols


References

MitoPedia concepts: SUIT protocol, Recommended 


MitoPedia methods: Respirometry