SUIT-018 AmR ce-pce D068: Difference between revisions
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|info='''A''' -'''[[SUIT-018]]''' | |info='''A''' -'''[[SUIT-018]]''' | ||
|application=AmR | |application=AmR | ||
|SUIT number= | |SUIT number=D068_1GMS;2D;3Ama | ||
}} | }} | ||
::: '''[[MitoPedia: SUIT]]''' - 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 concentration. ย | ::: '''[[MitoPedia: SUIT]]''' - 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 concentration. ย | ||
::: '''[[SUIT protocol pattern]]:''' | ::: '''[[SUIT protocol pattern]]:''' 1GMS;2D- | ||
SUIT-018 AmR ce-pce D068 is aย protocol to study simultaneously the oxygen dependence of O<sub>2</sub> flux and H<sub>2</sub>O<sub>2</sub> production on permeabilized cells at various oxygen concentrations. The oxygen concentration in the chamber can be altered injecting nitrogen with a syringe. To test the inhibitory effect of the AmR assay, [[DTPA]], [[SOD]], [[HRP]] and [[Amplex UltraRed|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]]. | SUIT-018 AmR ce-pce D068 is aย protocol to study simultaneously the oxygen dependence of O<sub>2</sub> flux and H<sub>2</sub>O<sub>2</sub> production on permeabilized cells at various oxygen concentrations. The oxygen concentration in the chamber can be altered injecting nitrogen with a syringe. To test the inhibitory effect of the AmR assay, [[DTPA]], [[SOD]], [[HRP]] and [[Amplex UltraRed|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]]. | ||
__TOC__ | __TOC__ | ||
ย Communicated byย [[Komlodi T]] and [[Gnaiger E]] (last update 2020-02- | ย Communicated byย [[Komlodi T]] and [[Gnaiger E]] (last update 2020-02-25) | ||
== Representative traces == | == Representative traces == | ||
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[[File:SUIT-018 AmR ce-pce D068 AmR trace.png|600px]] | [[File:SUIT-018 AmR ce-pce D068 AmR trace.png|600px]] | ||
{{Template: SUIT-018 | {{Template:SUIT-018 D068}} | ||
== Strengths and limitations == | == Strengths and limitations == | ||
:::+ Simple protocol to evaluate the oxygen dependence of H<sub>2</sub>O<sub>2</sub> production in LEAK and OXPHOS | :::+ Simple protocol to evaluate the oxygen dependence of H<sub>2</sub>O<sub>2</sub> production in LEAK and OXPHOS. | ||
ย | :::- Measurements with Amplex UltraRed assay cannot be carried out with liver homogenate. | ||
::: | |||
:::- Reoxygenation and nitrogen injection could lead to bubble formation in the O2k-Chamber. | :::- 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. | :::- CIV activity and cytochrome ''c'' test cannot be performed together with the fluorescence. | ||
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== 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 (isolated mitochondria, tissue homogenate and permeabilized cells) changing the oxygen concentration in the same protocol. | :::* [[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 (isolated mitochondria, tissue homogenate and permeabilized cells) changing the oxygen concentration in the same protocol. | ||
:::* [[SUIT-009 | :::* [[SUIT-009]] to investigate H<sub>2</sub>O<sub>2</sub> production driven by the [[Reverse electron flow from CII to CI| reverse electron transfer]] (RET) in isolated mitochondria, tissue homogenate and permeabilized cells. | ||
== References == | == References == |
Revision as of 16:48, 26 February 2020
Description
Abbreviation: O2 dependence-H2O2
Reference: A -SUIT-018
SUIT number: D068_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: 1GMS;2D-
SUIT-018 AmR ce-pce D068 is a protocol to study simultaneously the oxygen dependence of O2 flux and H2O2 production on permeabilized cells at various oxygen concentrations. The oxygen concentration in the chamber can be altered 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.
Communicated by Komlodi T and Gnaiger E (last update 2020-02-25)
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.
- - Measurements with Amplex UltraRed assay cannot be carried out with liver homogenate.
- - 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
- SUIT-018 AmR mt D041 for simultaneous determination of O2 and H2O2 flux in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells) changing the oxygen concentration in the same protocol.
- SUIT-009 to investigate H2O2 production driven by the reverse electron transfer (RET) in isolated mitochondria, tissue homogenate and permeabilized cells.
References
Year | Reference | Organism | Tissue;cell | |
---|---|---|---|---|
MiPNet24.10 H2O2 flux analysis | 2021-10-22 | Hydrogen peroxide flux analysis using Amplex UltraRed assay in MiR05-Kit with DatLab 7.4 | ||
Komlodi 2021 BEC AmR-O2 | 2021 | Komlรณdi T, Sobotka O, Gnaiger E (2021) Facts and artefacts on the oxygen dependence of hydrogen peroxide production using Amplex UltraRed. Bioenerg Commun 2021.4. https://doi.org/10.26124/bec:2021-0004 | Saccharomyces cerevisiae | Other cell lines |
MiPNet20.14 AmplexRed H2O2-production | 2019-06-24 | O2k-FluoRespirometry: HRR and simultaneous determination of H2O2 production with Amplex UltraRed. | Mouse | Heart |
Komlodi 2018 Methods Mol Biol | 2018 | Komlodi T, Sobotka O, Krumschnabel G, Bezuidenhout N, Hiller E, Doerrier C, Gnaiger E (2018) Comparison of mitochondrial incubation media for measurement of respiration and hydrogen peroxide production. Methods Mol Biol 1782:137-55. | Human Mouse | Skeletal muscle HEK |
Makrecka-Kuka 2015 Biomolecules | 2015 | Makrecka-Kuka M, Krumschnabel G, Gnaiger E (2015) High-resolution respirometry for simultaneous measurement of oxygen and hydrogen peroxide fluxes in permeabilized cells, tissue homogenate and isolated mitochondria. https://doi.org/10.3390/biom5031319 | Human Mouse | Heart Nervous system HEK |
Krumschnabel 2015 Methods Mol Biol | 2015 | Krumschnabel G, Fontana-Ayoub M, Sumbalova Z, Heidler J, Gauper K, Fasching M, Gnaiger E (2015) Simultaneous high-resolution measurement of mitochondrial respiration and hydrogen peroxide production. Methods Mol Biol 1264:245-61. | Mouse | Nervous system |
MitoPedia concepts: SUIT protocol, SUIT A, Find
MitoPedia methods:
Fluorometry