SUIT-006 O2 ce-pce D029

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SUIT-006 O2 ce-pce D029

Description

Ce1;1Dig;1PM;2D;2c;3Omy;4U;5Ama.png

Abbreviation: CCP ce-pce PM

Reference: A to analyse the coupling control on permeabilized cells in N(PM) pathway control state- SUIT-006

SUIT number: D029_ce1;1Dig;1PM;2D;2c;3Omy;4U;5Ama

O2k-Application: O2

MitoPedia: SUIT
SUIT protocol pattern: ce1;1Dig;1PM;2D;2c;3Omy;4U;5Ama

SUIT-006 O2 ce-pce D029 is a coupling control protocol for permeabilized cells. Living cells are added in the chamber, allowing to analyse ROUTINE respiration. The cells are further permeabilized inside the O2k chamber, and different coupling control states are assessed (L(n) - P - L(Omy) - E) at N(PM) pathway control state. The use of oligomycin is optional, however, it provides important information when residual and endogenous adenylates are present (which may happen if ATPases are active in the sample). This situation may lead to overestimated LEAK respiration measured in the absence of adenylates - L(n). Therefore, oligomycin can be used to verify whether this occurs and obtain the LEAK state appropriately.

Communicated by Cardoso LHD, Iglesias-Gonzalez J, Komlodi T and Doerrier C (last update 2019-06-06) 

Representative traces

SUIT-006 O2 pce D029 traces.png

MitoPedia: SUIT

Steps and respiratory states

Ce1;1Dig;1PM;2D;2c;3Omy;4U;5Ama.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 ROX 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
  • NADH-linked substrates (type N-pathway to Q).
  • OXPHOS capacity, P (with saturating [ADP]), active OXPHOS state.
  • Addition of cytochrome c yields a test for integrity of the mtOM. Stimulation by added cytochrome c would indicate an injury of the mtOM and limitation of respiration in the preceding state without added c due to loss of cytochrome c. Typically, cytochrome c is added immediately after the earliest ADP-activation step (P, OXPHOS-capacity with saturating [ADP]).
3Omy PMLOmy N CI 1PM;2D;2c;3Omy
  • NADH-linked substrates (type N-pathway to Q).
  • Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(Omy), after blocking the ATP synthase with oligomycin.
  • The addition of oligomycin is optional depending on the experimental question to resolve. If it is known that Ln has the same values for LOmy for a kind of sample and substrates, this step may be skipped. If the carrier of oligomycin (EtOH) is used instead, it is possible to evaluate whether it affects respiration. In these cases, LEAK state is not reached again.
4U PME N CI 1PM;2D;2c;3Omy;4U
5Ama ROX 1PM;2D;2c;3Omy;4U;5Ama
  • 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

  • This protocol is indicated when the aim is to analyse coupling control in permeabilized cells in NADH-pathway using PM as substrates.
+ Reasonable duration of the experiment.
+ This protocol can be extended with the Complex IV module.
- This protocol analyses the coupling control in NADH-pathway (with PM), however, it is less specific than a similar protocol containing the complex II inhibitor malonic acid, that would allow to analyse the NADH-pathway without any contribution of the S-pathway.
- This protocol does not include internal ET-pathway control steps.
- Careful washing is required after the experiment to avoid carry-over of uncoupler and inhibitors, if used.


Compare SUIT protocols

  • SUIT-001 O2 ce-pce D003: Extended protocol with SUIT-006 as the initial coupling control module, for permeabilized cells.
  • SUIT-003: Coupling control protocol for non-permeabilized cells (ce), counterpart to SUIT-006 for mt-preparations (mtprep).


References

MitoPedia concepts: SUIT protocol, SUIT A, Find 


MitoPedia methods: Respirometry