SUIT-009 O2 ce-pce D016

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SUIT-009 O2 ce-pce D016

Description

SUIT-009


Reference: B - SUIT-009

SUIT number: D016_ce1;1Dig;1S;2D;3P;4Rot;5Ama

MitoPedia: SUIT - short protocol for mt-respiration in ce: non-permeabilized cells, pce: permeabilized cells.
SUIT protocol pattern: 1S;2D;3P;4Rot-

This is a short protocol to investigate O2 flux in permeabilized cells (non-permeabilized cells must be added to the chambers and then permeabilized as a part of the protocol). It is recommended as a control experiment for SUIT-009 AmR ce-pce D019 without using the Amplex UltraRed assay in order to detect the effect of the fluorescence dye on the O2 flux and with the cytochrome c to assess the integrity of the mitochondrial outer membrane. We do not recommend this protocol for determination of the O2 flux in the absence of Rotenone. The following Coupling control protocol can be used to assess Succinate-linked respiration with Rotenone: SUIT-006 O2 mt D022.

Communicated by Doerrier C, Huete-Ortega M, Iglesias-Gonzalez J, Komlodi T and Gnaiger E (last update 2019-06-06)
MitoPedia: SUIT

Steps and respiratory states

SUIT-009


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.
2Dig Dig ce1;1Dig
  • Optimum effective digitonin concentration for complete plasma membrane permeabilization.


Step State Pathway Q-junction Comment - Events (E) and Marks (M)
1S SL(n) S CII 1S
2D SP S CII 1S;2D
2c ScP S CII 1S;2D;2c
  • Succinate, S ( type S-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]).
3P NSP NS CI&II 1S;2D;3P
  • 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 1S;2D;3P;4Rot
5Ama ROX 1S;2D;3P;4Rot;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).


Step Respiratory state Pathway control ET-Complex entry into Q-junction Comment
## Tm TmE CIV CIV
## Azd ROX


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Strengths and limitations

  • We do not recommend this protocol to assess just respiration since rotenone is not added at the beginning and does not prevent the inhibitory effect of oxaloacetate on the succinate dehydrogenase. This protocol can be used as a control to investigate the effect of the fluorescence dyes on the respiration.
  • It is a protocol to analyse the S- and NS-pathway linked respiration.
  • For H2O2-Application, choose SUIT-009 AmR ce-pce D019.
+ Cytochrome c test can be performed in the S-OXPHOS state.
+ Short duration of the experiment.
+ This protocol can be extended with the Complex IV module.
- A succinate concentration > 10 mM might be required to reach S-OXPHOS capacity.
- In some cell lines e.g. HEK293T, P does not have any effect because of the downregulation of the N-linked pathway.

Compare SUIT protocols

SUIT-006 O2 mt D022: to investigate the S(Rot)-linked respiration.

References

MitoPedia concepts: SUIT protocol, SUIT B, Find 


MitoPedia methods: Respirometry