Description
Abbreviation: NS(P)
Reference: A: SUIT-018 to study simultaneously the oxygen dependence of O2 flux and H2O2 production in mitochondrial preparations (isolated mitochondria and tissue homogenate).
SUIT number: D040_1GMS;2D;3Ama
O2k-Application: AmR
- SUIT-category: NS(GM)
- SUIT protocol pattern: orthogonal 1GMS;2D;3Ama
This is a short protocol to study simultaneously the oxygen dependence of O2 flux and H2O2 production on isolated mitochondria or tissue homogenate (except of liver homogenate). You can run the protocol 1.)under tissue normoxia (50-60 Β΅M = [O2]) SUIT-018 AmR mt D031, 2.) under tissue hyperoxia [> 120 Β΅M = [O2]] SUIT-018 AmR mt D040 or 3.) you can change the oxygen concentration during the experiment SUIT-018 AmR mt D041. The oxygen concentration in the chamber can be changed by the injection of nitrogen with a syringe. It is recommended to run both SUIT-018 AmR mt D031 and SUIT-018 AmR mt D040 in paralell or SUIT-018 AmR mt D041 alone in order to test the H2O2 production at different oxygen concentrations.
Communicated by [Komlodi T]] (last update 2019-02-01)
References
Steps and respiratory states
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
0DTPA |
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0SOD |
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0HRP |
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0AmR |
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Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
1GMS | GMSL(n) | NS | CI&CII | 1GMS
|
2D | GMSP | NS | CI&CII | 1GMS;2D
|
3Ama | ROX | 1GMS;2D;3Ama
|
Strengths and limitations
- + Reasonable duration of the experiment.
- + Simple protocol to evaluate the oxygen dependence of H2O2 production.
- - Measurements with Amplex UltraRed assay cannot be carried out with liver homogenate.
- - H2O2 production can be studied in two different states, LEAK and OXPHOS.
- - Reoxygenation and nitrogen injection could lead to bubble formation in the O2k-Chamber
- - CIV activity and Cytochrome c test cannot be performed together with the fluorescence.
Compare SUIT protocols
MitoPedia concepts: SUIT protocol, SUIT A, Find
MitoPedia methods:
Fluorometry