SUIT-018 AmR ce-pce D068: Difference between revisions
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{{MitoPedia | {{MitoPedia | ||
|abbr=O<sub>2</sub> dependence-H<sub>2</sub>O<sub>2</sub> | |abbr=O<sub>2</sub> dependence-H<sub>2</sub>O<sub>2</sub> | ||
|description=[[File:Ce1;1Dig; | |description=[[File:Ce1;1Dig;2GMS;3D;4Ama.png|290px]] | ||
|info='''A''' -'''[[SUIT-018]]''' | |info='''A''' -'''[[SUIT-018]]''' - SUIT protocol for simultaneous determination of O<sub>2</sub> and H<sub>2</sub>O<sub>2</sub> flux in permeabilized cells at various oxygen concentrations | ||
|application=AmR | |application=AmR | ||
|SUIT number=D068_ce1;1Dig;1GMS;2D;3Ama | |SUIT number=D068_ce1;1Dig;1GMS;2D;3Ama | ||
}} | }} | ||
SUIT-018 AmR ce-pce D068 is a protocol to simultaneously study the oxygen dependence of O<sub>2</sub> flux and H<sub>2</sub>O<sub>2</sub> flux in permeabilized cells, and to test the chemical inhibitory effect of the AmR assay ([[SOD]], [[HRP]] and [[Amplex UltraRed | AmR]]) on ROUTINE-respiration of living cells. 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 [[Amplex UltraRed|AmR]] are added in ROUTINE. However, 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. To decrease or increase the oxygen concentration in the O2k-Chamber, see [[Setting the oxygen concentration]]. | |||
SUIT-018 AmR ce-pce D068 is a protocol to study | |||
__TOC__ | __TOC__ | ||
Communicated by [[Komlodi T]] and [[Gnaiger E]] (last update 2020- | Communicated by [[Komlodi T]], [[Cecatto C]] and [[Gnaiger E]] (last update 2020-06-09) | ||
== Representative traces == | == Representative traces == | ||
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== Strengths and limitations == | == Strengths and limitations == | ||
:::+ Simple protocol to evaluate the oxygen dependence of H<sub>2</sub>O<sub>2</sub> | :::+ Simple protocol to evaluate the oxygen dependence of H<sub>2</sub>O<sub>2</sub> flux in [[LEAK respiration | LEAK]] and [[Oxidative_phosphorylation |OXPHOS]]. | ||
:::+ Inhibitory effect of the AmR assay can be tested in the same protocol | :::+ Inhibitory effect of the AmR assay can be tested in the same protocol on [[ROUTINE-respiration | ROUTINE]] respiration. | ||
:::- Reoxygenation and nitrogen injection | :::- Reoxygenation and nitrogen injection can lead to bubble formation in the O2k-Chamber. | ||
:::- CIV activity and cytochrome ''c'' test cannot be performed together with the fluorescence. | :::- CIV activity and cytochrome ''c'' test cannot be performed together with the fluorescence. | ||
== Compare SUIT protocols == | == Compare SUIT protocols == | ||
:::* [[SUIT-018 AmR mt D041]] for simultaneous determination of O<sub>2</sub> and H<sub>2</sub>O<sub>2</sub> flux in [[Mitochondrial preparations| mitochondrial preparations]] such as isolated mitochondria, tissue homogenate and permeabilized cells at different oxygen | :::* [[SUIT-018 AmR mt D041]] for simultaneous determination of O<sub>2</sub> and H<sub>2</sub>O<sub>2</sub> flux in [[Mitochondrial preparations| mitochondrial preparations]] such as isolated mitochondria, tissue homogenate and permeabilized cells at different oxygen concentrations in the same protocol. | ||
::: | |||
== Chemicals and syringes == | |||
{{Template:Chemicals AmR}} | |||
{{Template:Chemicals ce}} | |||
{{Template:Chemicals SUIT-018}} | |||
::: Suggested stock concentrations are shown in the specific DL-Protocol. | |||
== References == | == References == |
Latest revision as of 12:06, 7 March 2024
Description
Abbreviation: O2 dependence-H2O2
Reference: A -SUIT-018 - SUIT protocol for simultaneous determination of O2 and H2O2 flux in permeabilized cells at various oxygen concentrations
SUIT number: D068_ce1;1Dig;1GMS;2D;3Ama
O2k-Application: AmR
SUIT-018 AmR ce-pce D068 is a protocol to simultaneously study the oxygen dependence of O2 flux and H2O2 flux in permeabilized cells, and to test the chemical inhibitory effect of the AmR assay (SOD, HRP and AmR) on ROUTINE-respiration of living cells. 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 are added in ROUTINE. However, 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. To decrease or increase the oxygen concentration in the O2k-Chamber, see Setting the oxygen concentration.
Communicated by Komlodi T, Cecatto C and Gnaiger E (last update 2020-06-09)
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 flux in LEAK and OXPHOS.
- + Inhibitory effect of the AmR assay can be tested in the same protocol on ROUTINE respiration.
- - Reoxygenation and nitrogen injection can lead to bubble 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 concentrations in the same protocol.
Chemicals and syringes
Step | Chemical(s) and link(s) | Comments |
---|---|---|
H2O2 | Hydrogen Peroxide (H2O2) |
Step | Chemical(s) and link(s) | Comments |
---|---|---|
0DTPA | DTPA | This step can be skipped |
0SOD | Superoxide Dismutase (SOD) | |
0HRP | Horseradish peroxidase (HRP) | |
0AmR | Amplex UltraRed (AmR) |
Step | Chemical(s) and link(s) | Comments |
---|---|---|
1Dig | Digitonin (Dig) |
Step | Chemical(s) and link(s) | Comments |
---|---|---|
1GMS | Glutamate (G), Malate (M) and Succinate (S) | |
2D | ADP (D) | |
3Ama | Antimycin A (Ama) |
- Suggested stock concentrations are shown in the specific DL-Protocol.
References
MitoPedia concepts: SUIT protocol, SUIT A, Find
MitoPedia methods:
Fluorometry