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Difference between revisions of "SUIT-009 AmR ce-pce D019"

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== 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.

Revision as of 12:08, 21 January 2019


high-resolution terminology - matching measurements at high-resolution


SUIT-009 AmR ce-pce D019

Description

SUIT9

Abbreviation: NS(P)

Reference: A: SUIT-009 short protocol for H2O2 (AmR) in pce.

SUIT number: D019_1S;2D;3P;4Rot;5Ama

O2k-Application: AmR

SUIT-category: NS(P)
SUIT protocol pattern: orthogonal 1S;2D;3P;4Rot
SUIT-9 AmR pce D19 Overview.png

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 UltraRedยฎ 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 ยฐC.


MitoPedia concepts: SUIT protocol, SUIT A, Find 


MitoPedia methods: Fluorometry 

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.