SUIT-018 O2 mt D054

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Bioblasts - Richard Altmann and MiPArt by Odra Noel
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SUIT-018 O2 mt D054

Description

1GMS;2D;2c;3Ama.png


Reference: B: SUIT-018

SUIT number: D054_1GMS;2D;3Ama

O2k-Application: O2


SUIT-018 O2 mt D054 is a short protocol to study the influence of a fluorescence dye on the O2 flux. During this protocol the [O2] can be modified by injecting nitrogen in the chamber.

Communicated by Komlodi T and Gnaiger E  (last update 2019-09-24)


Representative traces

SUIT-018 O2 mt D054.png

MitoPedia: SUIT

Steps and respiratory states

SUIT-018

Step State Pathway Q-junction Comment - Events (E) and Marks (M)
1GMS GMSL(n) NS CI&CII 1GMS
  • Respiratory stimulation by simultaneous action of type N substrates & succinate, with convergent electron flow in the NS-pathway for reconstitution of TCA cycle function.
  • Non-phosphorylating resting state (LEAK state); LEAK respiration L(n) in the absence of ADP, ATP, AMP (no adenylates).
2D GMSP NS CI&CII 1GMS;2D
  • 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.
2c GMScP NS CI&CII 1GMS;2D;2c
  • 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.
  • Addition of cytochrome c yields a test for integrity of the mtOM (cytochrome c control efficiency). 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 (OXPHOS capacity P with saturating [ADP]).
3Ama ROX 1GMS;2D;2c;3Ama
  • 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
## AsTm AsTmE CIV CIV
## Azd ROX


Questions.jpg


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Strengths and limitations

+ Reasonable duration of the experiment.
- CIV activity and cytochrome c test can be performed.

Compare SUIT protocols

Chemicals and syringes

Step Chemical(s) and link(s) Comments
1GMS Glutamate (G), Malate (M) and Succinate (S)
2D ADP (D)
2c Cytochrome c (c)
3Ama Antimycin A (Ama)
Suggested stock concentrations are shown in the specific DL-Protocol.



MitoPedia concepts: SUIT protocol, SUIT B, Find 


MitoPedia methods: Respirometry