SUIT-022

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SUIT-022

Description

Ce1;ce2KCN;ce3SHAM.v2.png

Abbreviation: AOX (ce CN+SHAM)

Reference: A: Determination of the respiration due to the alternative oxidase pathway

SUIT protocol pattern: ce1;ce2KCN;ce3SHAM

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

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

Specific SUIT protocols

SUIT-022 O2 ce D051

Ce1;ce2KCN;ce3SHAM.v2.png Ce1;ce2KCN;ce3SHAM.png D051 O2 traces.png

MitoPedia: SUIT

Steps and respiratory states

Ce1;ce2KCN;ce3SHAM.v2.png Ce1;ce2KCN;ce3SHAM.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.
ce2KCN ROUTINE or ROX ce1;ce2KCN
  • Cyanide (usually added as KCN) is a competitive inhibitor of cytochrome c oxidase (CIV). In the presence of an active alternative oxidase (AOX) pathway, after the inhibition of CIV, cells remain in the physiological coupling state ROUTINE. In those occasions where the AOX is not active, after the inhibition of CIV, the (ROX state) is normally achieved, where Rox is the residual oxygen consumption due to oxidative side reactions estimated either after inhibition of CIII, 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. Cyanide inhibition is reversed by pyruvate and high oxygen levels.
ce3SHAM ROX ce1;ce2KCN;ce3SHAM


Questions.jpg


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Strengths and limitations

  • SUIT-022 provides an estimation of the cyanide-and antimycin A-resistant mitochondrial respiration due to the activity of alternative oxidase in cells on ROUTINE. To complement the study with the estimation of mitochondrial respiration due to the CIII-CIV pathway, it must be carried out in parallel to SUIT-023.
+ This protocol can be used with many types of cells, although optimal concentrations of inhibitors have to be determined.
+ 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: MiP concept, SUIT protocol, Recommended 


MitoPedia methods: Respirometry