Difference between revisions of "SUIT-018 AmR mt D031"
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{{MitoPedia | {{MitoPedia | ||
|abbr= | |abbr=O<sub>2</sub> dependence-H<sub>2</sub>O<sub>2</sub> | ||
|description=[[File:1GMS;2D;3Ama.png| | |description=[[File:1GMS;2D;3Ama.png|290px]] | ||
|info='''A''' | |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 mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells) at low oxygen concentration (tissue normoxia) | ||
|application=AmR | |application=AmR | ||
|SUIT number= | |SUIT number=D031_mt;1GMS;2D;3Ama | ||
}} | }} | ||
SUIT-018 AmR mt D031 is a short protocol to study simultaneously the oxygen dependence of O<sub>2</sub> flux and H<sub>2</sub>O<sub>2</sub> production on mitochondrial preparations. Is it possible to run the protocol under different oxygen concentration conditions: 1.) under tissue normoxia (50-60 µM = [O<sub>2</sub>]), 2.) under tissue hyperoxia (> 120 µM = [O<sub>2</sub>]) or 3.) you can change the oxygen concentration during the experiment. To decrease or increase the oxygen concentration in the O2k-Chamber, see [[Setting the oxygen concentration]]. We recommend to run [[SUIT-018 AmR mt D040]] simultaneously at high oxygen concentration as a control. | |||
SUIT-018 AmR mt D031 is a short protocol to study simultaneously the oxygen dependence of O<sub>2</sub> flux and H<sub>2</sub>O<sub>2</sub> production on | |||
__TOC__ | __TOC__ | ||
Communicated by | Communicated by [[Komlodi T]], [[Gnaiger E]] (last update 2019-09-24) | ||
== Representative traces == | |||
[[File:SUIT-018 mt AmR D031 O2 DL-Files.png|600px]] | |||
[[File:SUIT-018 AmR mt D031 DL-Files.png|600px]] | |||
{{Template: SUIT-018 Specific SUIT}} | {{Template: SUIT-018 Specific SUIT}} | ||
== Strengths and limitations == | == Strengths and limitations == | ||
:::* [[SUIT-018 AmR mt D040]] should be | :::* [[SUIT-018 AmR mt D040]] should be run in parallel (tissue hyperoxia condition). | ||
:::+ Reasonable duration of the experiment. | :::+ Reasonable duration of the experiment. | ||
:::+ 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. | ||
Line 24: | Line 28: | ||
== 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 | :::* [[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-006 AmR mt D048]] to investigate the dependence of H<sub>2</sub>O<sub>2</sub> flux on the mt-membrane | :::* [[SUIT-006 AmR mt D048]] to investigate the dependence of H<sub>2</sub>O<sub>2</sub> flux on the mt-membrane potential on the N-control state in mitochondrial preparations. | ||
:::* [[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) | :::* [[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. | ||
== Chemicals and syringes == | |||
{{Template:Chemicals AmR}} | |||
{{Template:Chemicals SUIT-018}} | |||
::: Suggested stock concentrations are shown in the specific DL-Protocol. | |||
== References == | == References == |
Latest revision as of 12:09, 7 March 2024
Description
Abbreviation: O2 dependence-H2O2
Reference: A -SUIT-018 - SUIT protocol for simultaneous determination of O2 and H2O2 flux in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells) at low oxygen concentration (tissue normoxia)
SUIT number: D031_mt;1GMS;2D;3Ama
O2k-Application: AmR
SUIT-018 AmR mt D031 is a short protocol to study simultaneously the oxygen dependence of O2 flux and H2O2 production on mitochondrial preparations. Is it possible to run the protocol under different oxygen concentration conditions: 1.) under tissue normoxia (50-60 µM = [O2]), 2.) under tissue hyperoxia (> 120 µM = [O2]) or 3.) you can change the oxygen concentration during the experiment. To decrease or increase the oxygen concentration in the O2k-Chamber, see Setting the oxygen concentration. We recommend to run SUIT-018 AmR mt D040 simultaneously at high oxygen concentration as a control.
Communicated by Komlodi T, Gnaiger E (last update 2019-09-24)
Representative traces
Steps and respiratory states
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) |
---|---|---|---|---|
1GMS | GMSL(n) | NS | CI&CII | 1GMS
|
2D | GMSP | NS | CI&CII | 1GMS;2D
|
3Ama | ROX | 1GMS;2D;3Ama
|
Strengths and limitations
- SUIT-018 AmR mt D040 should be run in parallel (tissue hyperoxia condition).
- + Reasonable duration of the experiment.
- + 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-006 AmR mt D048 to investigate the dependence of H2O2 flux on the mt-membrane potential on the N-control state in mitochondrial preparations.
- SUIT-009 to investigate H2O2 production driven by the reverse electron transfer (RET) in isolated mitochondria, tissue homogenate and permeabilized cells.
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 |
---|---|---|
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
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 | Komlódi 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