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The renal tissue obtained a significantly higher production of ROS as shown by DCFH levels in both exposed groups.
In the present study, quantification of intracellular ROS production was conducted using DCFH as fluorescent probe.
(a) ROS levels by DCFH method; (b) lipid peroxidation by TBARS (thiobarbituric acid reactive substance); (c) glutathione levels (GSH); (d) total antioxidant capacity by FRAP method.
Upon cellular uptake, the DCFH-DA probe is hydrolyzed to DCFH by intracellular esterases and oxidized to highly uorescent dichlorouorescein (DCF) in the presence of ROS.
Abbreviations: DCFH, 2',7'-dichlorodihydrofluorescein; DCFH-DA.
esterase (Boehringer Mannheim, Germany) and incubated at 25degC for 15 min to yield dichloroflurescein (DCFH) after deacetylation.
The dried sprouts were ground in a hammer mill (Culatti, Model: JKA Werk, Type: DCFH, Germany) and sieved with a 60 mesh screen (Model BS 410, Endeco London, England).
In healthy cells, DCFH-DA crosses cell membranes and is enzymatically hydrolyzed by intracellular esterases to non-fluorescent DCFH. In the presence of ROS, DCFH is oxidized to highly fluorescent di-chlorofluorescein (DCF).
Nonfluorescent HE is oxidized to fluorescent 2-hydroxyethidium by [O.sub.2]*, whereas DCFH is oxidized to dichiorofluorescein (DCF) by [H.sub.2][O.sub.2] and peroxidases (Rothe and Valet 1990).
Oxidation of DCFH within cells leads to fluorescent dichlorofluorescein, which can easily be visualized (strong emission at 525 nm with excitation at 488 nm).
* Tube B: cells + DCFH (2' 7' dichlorofluorescein) evaluation of basal oxidative burst activity;
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