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SUIT-031 O2 ce-pce D079

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SUIT-031 O2 ce-pce D079

Description

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).




Questions.jpg


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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
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