Difference between revisions of "SUIT-009 AmR ce-pce D019"
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[[File:SUIT-9 AmR pce D19 Overview.png|right|600px]] | [[File:SUIT-9 AmR pce D19 Overview.png|right|600px]] | ||
== 1S;2D;3P;4Rot;5Ama-== | == 1S;2D;3P;4Rot;5Ama-== | ||
SUIT 9 is a short protocol for simultaneous determination of O<sub>2</sub> flux and the rate of [[MiPNet20.14 AmplexRed H2O2-production|H<sub>2</sub>O<sub>2</sub> production]] (H<sub>2</sub>O<sub>2</sub> flux) on isolated mitochondria,tissue homogenate (except of liver) and permeabilized cells. Succinate (S) supports the reverse electron transfer (RET) initiated H<sub>2</sub>O<sub>2</sub> production in the LEAK state. Addition of ADP decreases H<sub>2</sub>O<sub>2</sub> flux due to depolarization of the mitochondrial membrane. Pyruvate (P) supports [[NADH Electron transfer-pathway state|NADH-pathway (N)]], and usually in the presence of ADP it does not increase further the H<sub>2</sub>O<sub>2</sub> flux. Antimycin A (Ama) inhibits Complex III (CIII) and results in the elevation of the H<sub>2</sub>O<sub>2</sub> flux. The sensitivity of the Amplex UltraRed® assay (for determining H<sub>2</sub>O<sub>2</sub> production) is changing over the experimental time and upon addition of chemicals. To correct H<sub>2</sub>O<sub>2</sub> flux for the sensitivity changes several [[MiPNet20.14_AmplexRed_H2O2-production#Calibration_with_H2O2|H<sub>2</sub>O<sub>2</sub> calibration steps]] are done during the experiment. The measurement is carried out in MiR05-Kit at 37 °C. | SUIT 9 is a short protocol for simultaneous determination of O<sub>2</sub> flux and the rate of [[MiPNet20.14 AmplexRed H2O2-production|H<sub>2</sub>O<sub>2</sub> production]] (H<sub>2</sub>O<sub>2</sub> flux) on isolated mitochondria,tissue homogenate (except of liver) and permeabilized cells. Succinate (S) supports the reverse electron transfer (RET) initiated H<sub>2</sub>O<sub>2</sub> production in the LEAK state. Addition of ADP decreases H<sub>2</sub>O<sub>2</sub> flux due to depolarization of the mitochondrial membrane. Pyruvate (P) supports [[NADH Electron transfer-pathway state|NADH-pathway (N)]], and usually in the presence of ADP it does not increase further the H<sub>2</sub>O<sub>2</sub> flux. Antimycin A (Ama) inhibits Complex III (CIII) and results in the elevation of the H<sub>2</sub>O<sub>2</sub> flux. The sensitivity of the Amplex UltraRed® assay (for determining H<sub>2</sub>O<sub>2</sub> production) is changing over the experimental time and upon addition of chemicals. To correct H<sub>2</sub>O<sub>2</sub> flux for the sensitivity changes several [[MiPNet20.14_AmplexRed_H2O2-production#Calibration_with_H2O2|H<sub>2</sub>O<sub>2</sub> calibration steps]] are done during the experiment. The measurement is carried out in MiR05-Kit at 37 °C. | ||
== References == | |||
{{#ask:[[Category:Publications]] [[Additional label::SUIT-009 AmR pce D019]] [[Additional label::O2k-Protocol]] | |||
| ?Was published in year=Year | |||
| ?Has title=Reference | |||
| ?Mammal and model=Organism | |||
| ?Tissue and cell=Tissue;cell | |||
| format=broadtable | |||
| limit=5000 | |||
| offset=0 | |||
| sort=Was published in year | |||
| order=descending | |||
}} | |||
== Strengths and limitations == | |||
::: + You can investigate in the absence of ADP the reverse electron transfer supported H<sub>2</sub>O<sub>2</sub> production in S respiring mitochondria,which will be inhibited by ADP owing to the depolarisation of the mt-membrane. | |||
::: - CIV activity and Cytochrome c test cannot be performed together with the fluorescence. | |||
::: - The H<sub>2</sub>O<sub>2</sub> flux will be much lower compared to that observed with isolated mitochondria, which might be explained by the high antioxidant capacity of the cytosol. | |||
::: - In some cell lines e.g. HEK293T, P does not have any effect because of the downregulation of the N-linked pathway. | |||
{{MitoPedia concepts | {{MitoPedia concepts | ||
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|mitopedia method=Fluorometry | |mitopedia method=Fluorometry | ||
}} | }} | ||
Revision as of 17:06, 21 January 2019
Description
Abbreviation: NS(P)
Reference: A: SUIT-009 short protocol for H2O2 (AmR) in pce.
SUIT number: D019_ce1;1Dig;1S;2D;3P;4Rot;5Ama
- SUIT-category: NS(P)
- SUIT protocol pattern: orthogonal 1S;2D;3P;4Rot;5Ama
SUIT protocols
1S;2D;3P;4Rot;5Ama-
SUIT 9 is a short protocol for simultaneous determination of O2 flux and the rate of H2O2 production (H2O2 flux) on isolated mitochondria,tissue homogenate (except of liver) and permeabilized cells. Succinate (S) supports the reverse electron transfer (RET) initiated H2O2 production in the LEAK state. Addition of ADP decreases H2O2 flux due to depolarization of the mitochondrial membrane. Pyruvate (P) supports NADH-pathway (N), and usually in the presence of ADP it does not increase further the H2O2 flux. Antimycin A (Ama) inhibits Complex III (CIII) and results in the elevation of the H2O2 flux. The sensitivity of the Amplex UltraRed® assay (for determining H2O2 production) is changing over the experimental time and upon addition of chemicals. To correct H2O2 flux for the sensitivity changes several H2O2 calibration steps are done during the experiment. The measurement is carried out in MiR05-Kit at 37 °C.
References
Strengths and limitations
- + You can investigate in the absence of ADP the reverse electron transfer supported H2O2 production in S respiring mitochondria,which will be inhibited by ADP owing to the depolarisation of the mt-membrane.
- - CIV activity and Cytochrome c test cannot be performed together with the fluorescence.
- - The H2O2 flux will be much lower compared to that observed with isolated mitochondria, which might be explained by the high antioxidant capacity of the cytosol.
- - In some cell lines e.g. HEK293T, P does not have any effect because of the downregulation of the N-linked pathway.
MitoPedia concepts:
SUIT protocol,
SUIT A,
Find
MitoPedia methods:
Fluorometry