SUIT-018 AmR mt D040: Difference between revisions
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|info='''A''':ย 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)-'''[[SUIT-018]]''' | |info='''A''':ย 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)-'''[[SUIT-018]]''' | ||
|application=AmR | |application=AmR | ||
|SUIT number= | |SUIT number=D040_mt;1GMS;2D;3Ama | ||
}} | }} | ||
::: '''[[Categories of SUIT protocols |SUIT-category]]:''' NS(GM) | ::: '''[[Categories of SUIT protocols |SUIT-category]]:''' NS(GM) | ||
::: '''[[SUIT protocol pattern]]:''' | ::: '''[[SUIT protocol pattern]]:''' linear 1GMS;2D- | ||
SUIT-018 AmR mt D040 is aย protocol to study | SUIT-018 AmR mt D040 is aย protocol to simultaneously study the oxygen dependence of O<sub>2</sub> flux and H<sub>2</sub>O<sub>2</sub> flux in mitochondrial preparations. In experiments, H<sub>2</sub>O<sub>2</sub> flux shows linear dependence on oxygen concentration in LEAK, OXPHOS, ET states and in ROX state measured in [[MiR05-Kit]] medium. It is therefore recommended to vary the oxygen level in the O2k-Chamber several times during an experiment. It is also advisable to either detect H<sub>2</sub>O<sub>2</sub> flux at the same state for high (tissue hyperoxia; [O<sub>2</sub>]> ~100-110 ยตM) and low (tissue normoxia; [O<sub>2</sub>]< ~70-80 ยตM) oxygen concentration, or keep within tissue normoxia. A further recommendation is to reduce the oxygen concentration in the O2k-Chamber before the sample addition. To decrease or increase the oxygen concentration in the O2k-Chamber, see [[Setting the oxygen concentration]]. | ||
__TOC__ | __TOC__ | ||
ย Communicated by [[ | ย Communicated by [[Tindle-Solomon L]], [[Cecatto C]], [[Komlodi T]], [[Gnaiger E]] (last update 2020-06-25) | ||
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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> production in LEAK and OXPHOS states. | :::+ Simple protocol to evaluate the oxygen dependence of H<sub>2</sub>O<sub>2</sub> production in LEAK and OXPHOS states. | ||
:::+ Combination of N- and S-pathways is physiologically more relevant than using simple substrate to support a mitochondrial pathway. | :::+ Combination of N- and S-pathways is physiologically more relevant than using simple substrate to support a specific mitochondrial pathway. | ||
:::+ | :::+ Option at the end of the protocol to study the effect of oxygen concentration in the ROX state (in the presence of Ama) both on the O<sub>2</sub> and H<sub>2</sub>O<sub>2</sub> fluxes. | ||
:::- It is advisable to adjust the optimal sample concentration in the O2k-Chamber beforehand | :::- It is advisable to adjust the optimal sample concentration in the O2k-Chamber beforehand to avoid unnecessary reoxygenations (high sample concentration) or very long protocols (low sample concentration causing slower oxygen consumption in the O2k-Chamber and thus, delays in reaching the required oxygen concentrations). | ||
:::- Measurements with Amplex UltraRed assay cannot be carried out with liver homogenate. | :::- Measurements with Amplex UltraRed assay cannot be carried out with liver homogenate. | ||
ย | :::- Reoxygenation and nitrogen injection can lead to bubble formation in the O2k-Chamber. | ||
:::- Reoxygenation and nitrogen injection | |||
:::- 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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:::* [[SUIT-018 AmR ce-pce D068]] a protocol for simultaneous determination of O<sub>2</sub> and H<sub>2</sub>O<sub>2</sub> flux in permeabilized cells. | :::* [[SUIT-018 AmR ce-pce D068]] a protocol for simultaneous determination of O<sub>2</sub> and H<sub>2</sub>O<sub>2</sub> flux in permeabilized cells. | ||
:::* [[SUIT-006 AmR mt D048]] to investigate the dependence of H<sub>2</sub>O<sub>2</sub> flux on | :::* [[SUIT-006 AmR mt D048]] a protocol to investigate the dependence of H<sub>2</sub>O<sub>2</sub> flux on 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) in isolated mitochondria, tissue homogenate and permeabilized cells. | :::* [[SUIT-009]] a protocol 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. | ||
ย | |||
== Chemicals and syringes == | |||
{{Template:Chemicals AmR}} | |||
{{Template:Chemicals SUIT-018}} | |||
::: Suggested stock concentrations are shown in the specific DL-Protocol. | |||
== References == | == References == | ||
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}} | }} | ||
ย | {{MitoPedia concepts|mitopedia concept=SUIT protocol, SUIT A, Find}} | ||
{{MitoPedia concepts | {{MitoPedia methods|mitopedia method=Fluorometry}} | ||
|mitopedia concept=SUIT protocol, SUIT A, Find | |||
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{{MitoPedia methods | |||
|mitopedia method=Fluorometry | |||
Latest revision as of 12:09, 7 March 2024
Description
Abbreviation: NS(GM)
Reference: A: simultaneous determination of O2 and H2O2 flux in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells)-SUIT-018
SUIT number: D040_mt;1GMS;2D;3Ama
O2k-Application: AmR
- SUIT-category: NS(GM)
- SUIT protocol pattern: linear 1GMS;2D-
SUIT-018 AmR mt D040 is a protocol to simultaneously study the oxygen dependence of O2 flux and H2O2 flux in mitochondrial preparations. In experiments, H2O2 flux shows linear dependence on oxygen concentration in LEAK, OXPHOS, ET states and in ROX state measured in MiR05-Kit medium. It is therefore recommended to vary the oxygen level in the O2k-Chamber several times during an experiment. It is also advisable to either detect H2O2 flux at the same state for high (tissue hyperoxia; [O2]> ~100-110 ยตM) and low (tissue normoxia; [O2]< ~70-80 ยตM) oxygen concentration, or keep within tissue normoxia. A further recommendation is to reduce the oxygen concentration in the O2k-Chamber before the sample addition. To decrease or increase the oxygen concentration in the O2k-Chamber, see Setting the oxygen concentration.
Communicated by Tindle-Solomon L, Cecatto C, Komlodi T, Gnaiger E (last update 2020-06-25)
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
- + Simple protocol to evaluate the oxygen dependence of H2O2 production in LEAK and OXPHOS states.
- + Combination of N- and S-pathways is physiologically more relevant than using simple substrate to support a specific mitochondrial pathway.
- + Option at the end of the protocol to study the effect of oxygen concentration in the ROX state (in the presence of Ama) both on the O2 and H2O2 fluxes.
- - It is advisable to adjust the optimal sample concentration in the O2k-Chamber beforehand to avoid unnecessary reoxygenations (high sample concentration) or very long protocols (low sample concentration causing slower oxygen consumption in the O2k-Chamber and thus, delays in reaching the required oxygen concentrations).
- - Measurements with Amplex UltraRed assay cannot be carried out with liver homogenate.
- - 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 D041a simple protocol 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-018 AmR ce-pce D068 a protocol for simultaneous determination of O2 and H2O2 flux in permeabilized cells.
- SUIT-006 AmR mt D048 a protocol to investigate the dependence of H2O2 flux on mt-membrane potential on the N-control state in mitochondrial preparations.
- SUIT-009 a protocol 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 | 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