SUIT-009 O2 mt D015

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SUIT-009 O2 mt D015

Description

SUIT9

Abbreviation: NS(P)

Reference: B: short protocol for respiration in mitochondrial preparations; mt: isolated mitochondria, tissue homogenate and permeabilized cells- SUIT-009

SUIT number: D015_1S;2D;3P;4Rot;5Ama

MitoPedia: SUIT
SUIT-category: NS(P)
SUIT protocol pattern: orthogonal 1S;2D;3P;4Rot-

This is a short protocol for determination of O2 flux in isolated mitochondria, tissue homogenate and permeabilized cells (already permeabilized when they are added to the chamber). It is recommended as a control experiment for SUIT-009 AmR mt D021 without using the Amplex UltraRed assay in order to detect the effect of the fluorescent 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 Iglesias-Gonzalez J, Komlodi T and Gnaiger E (last update 2019-04-02)
MitoPedia: SUIT

Steps and respiratory states

1S;2D;2c;3P;4Rot;5Ama.png

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
  • 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


Questions.jpg


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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 the oxaloacetate on the succinate dehydrogenase. This protocol should be used as a control to investigate the effect of the fluorescence dyes on the respiration.
  • It is a protocol to investigate the S- and NS-pathway linked respiration.
  • For H2O2-Application, use SUIT-009 AmR mt D021.
+ Cytochrome c test can be performed in the S-OXPHOS state.
+ Short protocol.
+ This protocol can be extended with the Complex IV module.
+ Contribution of N-pathway to the NS can be investigated after the addition of Rot.
- A succinate concentration > 10 mM might be required to reach S-OXPHOS capacity.
- In some cell lines e.g. HEK293T, P has not any effect because of the downregulation of the N-linked pathway.

Compare SUIT protocols

References

MitoPedia concepts: SUIT protocol, SUIT B, Find 


MitoPedia methods: Respirometry