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SUIT-031 O2 ce-pce D079

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SUIT-031 O2 ce-pce D079

Description

Ce1;1Dig;1PM;2D;2c;3S;4Rot;5U;6Ama.png

Abbreviation: PM+S+Rot

Reference: A to analyse O2 flux in the NS- pathway control state in permeabilized cells- SUIT-031

SUIT number: D079_ce1;1Dig;1PM;2D;2c;3S;4Rot;5U;6Ama

O2k-Application: O2

This is a protocol to investigate oxygen flux N- and NS-pathway and S-pathway control state in the OXPHOS state and S-pathway control state in the ET state in permeabilized cells. SUIT-031 Q mt D072 and SUIT-031 Q ce-pce D074 are designed to measure simultaneously O2 flux and the Q-redox state in isolated mitochondria and permeabilized cells.

Communicated by Komlodi T (last update 2021-12-02) 

Representative traces

D079 O2 traces.png

MitoPedia: SUIT

Steps and respiratory states

Ce1;1Dig;1PM;2D;2c;3S;4Rot;5U;6Ama.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.
1Dig REN ce1;1Dig
  • Optimum effective digitonin concentration for complete plasma membrane permeabilization.
Step State Pathway Q-junction Comment - Events (E) and Marks (M)
1PM PML(n) N CI 1PM
2D PMP N CI 1PM;2D
2c PMcP N CI 1PM;2D;2c
3S PMSP NS CI&II 1PM;2D;2c;3S
  • Respiratory stimulation by simultaneous action of type N substrates & succinate, with convergent electron flow in the NS-pathway for reconstitution of TCA cycle function.
  • OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
4Rot SP S CII 1PM;2D;2c;3S;4Rot
5U SE N CII 1PM;2D;2c;3S;4Rot;5U
6Ama 1PM;2D;2c;3S;4Rot;5U;6Ama
  • Rox is the residual oxygen consumption in the ROX state, due to oxidative side reactions, estimated after addition of antimycin A (inhibitor of CIII). Rox is subtracted from oxygen flux as a baseline for all respiratory states, to obtain mitochondrial respiration (mt).




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Strengths and limitations

  • SUIT-031 O2 mt D075 in combination with SUIT-006 O2 mt D022 provides a common reference for comparison of respiratory control in a large variety of species, tissues and cell types. Both SUIT protocols provide a mitochondrial mapping which allows:
1. to obtain reference values.
2. to evaluate mitochondrial physiological diversity, generating a mt-database on comparative mitochondrial physiology.
3. to screen specific defects.
+ This protocol can be extended with the Complex IV module.
+ Reasonable duration of the experiment.
- Careful washing is required after the experiment to avoid carry-over of uncoupler and inhibitors.


Compare SUIT protocols

  • SUIT-001 O2 mt D001 is an extended coupling-control proton in the N-pathway and contribution of G-, S-, Oct- and Gp-pathways in the ET state for isolated mitochondria, tissue homogenate and cells permeabilized before addition to the O2k-chamber.
  • SUIT-004 is designed to provide a quick assessment of the linear coupling control (L- P- E) with NADH-linked substrates (PM) and the contribution of the S-pathway to the ET state (N, NS, S).
  • SUIT-006 O2 mt D047 is a coupling-control protocol in the N-pathway for isolated mitochondria, tissue homogenate and cells permeabilized before addition to the O2k-chamber.
  • SUIT-008 is designed to assess the additivity between the N- and S-pathway in the Q-junction, providing a physiologically relevant estimate of maximum mitochondrial respiratory capacity.


Chemicals and syringes

Step Chemical(s) and link(s) Comments
1Dig Digitonin (Dig)
Step Chemical(s) and link(s) Comments
1PM Pyruvate (P) and Malate (M)
2D ADP (D)
2c Cytochrome c (c)
3S Succinate (S)
4Rot Rotenone (Rot)
5U Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U) Can be substituted for other uncoupler.
6Ama Antimycin A (Ama)
Suggested stock concentrations are shown in the specific DL-Protocol.


References

 YearReferenceOrganismTissue;cell
MiPNet24.12 NextGen-O2k: Q-Module2021-10-29
O2k-Manual
NextGen-O2k: Q-Module manual


MitoPedia concepts: SUIT protocol, SUIT A, Find 


MitoPedia methods: Respirometry