Description
Abbreviation: O2 dependence-H2O2
Reference: A -SUIT-018
SUIT number: D068_ce1;1Dig;1GMS;2D;3Ama
O2k-Application: AmR
- MitoPedia: SUIT - SUIT protocol for simultaneous determination of O2 and H2O2 flux in permeabilized cells at various oxygen concentration.
- SUIT protocol pattern: ce1;1Dig;1GMS;2D-
SUIT-018 AmR ce-pce D068 is a protocol to study simultaneously the oxygen dependence of O2 flux and H2O2 production and to test the inhibitory effect of the AmR assay on permeabilized cells at various oxygen concentrations. The oxygen concentration in the chamber can be decreased by injecting nitrogen with a syringe. To test the inhibitory effect of the AmR assay, DTPA, SOD, HRP and AmR were added in ROUTINE. The inhibitory effect of the AmR assay can be tested in separate protocols: SUIT-003 AmR ce D058 and SUIT-003 AmR ce D059. For further information, see: Amplex UltraRed page.
Communicated by Komlodi T and Gnaiger E (last update 2020-02-26)
Representative traces
Steps and respiratory states
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
ce1 | ROUTINE | ce1
|
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
0DTPA |
| |||
0SOD |
| |||
0HRP |
| |||
0AmR |
|
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
1Dig | ROX | ce1;1Dig
| ||
1GMS | GMSL(n) | NS | CI&CII | ce1;1Dig;1GMS
|
2D | GMSP | NS | CI&CII | ce1;1Dig,1GMS;2D
|
3Ama | ROX | ce1;1Dig,1GMS;2D;3Ama
|
Strengths and limitations
- + Simple protocol to evaluate the oxygen dependence of H2O2 production in LEAK and OXPHOS.
- + Inhibitory effect of the AmR assay can be tested in the same protocol in ROUTINE respiration.
- - Reoxygenation and nitrogen injection could lead to bubbles formation in the O2k-Chamber.
- - CIV activity and cytochrome c test cannot be performed together with the fluorescence.
Compare SUIT protocols
- SUIT-018 AmR mt D041 for simultaneous determination of O2 and H2O2 flux in mitochondrial preparations such as isolated mitochondria, tissue homogenate and permeabilized cells at different oxygen concentration ranges in the same protocol.
- SUIT-009 AmR ce-pce D019 to investigate H2O2 production driven by the reverse electron transfer (RET) in permeabilized cells.
References
MitoPedia concepts: SUIT protocol, SUIT A, Find
MitoPedia methods:
Fluorometry