Standardization of a fluorometric assay for measuring oxidative stress in irradiated cells.

Abstract:

:The present study was undertaken to standardize a dichlorofluorescein (DCF) assay for measurement of radiation-induced oxidation of dichlorofluorescin (DCFH) substrate in MCF-10 cells. This assay was highly sensitive and capable of detecting increased DCFH oxidation in the cells exposed to gamma radiation at doses as low as 1.5 cGy with linear dose-response curves. However, the slope of the dose-response curves varied considerably from one experiment to another and was influenced by the fluorescent substrate concentration and cell density. To make the assay reproducible so that results obtained from different experiments could be compared, a series of conversion factors and equations have been established to normalize the data for these variables. The results demonstrate that the DCF assay, as standardized in the present study, is highly reproducible with acceptable assay precision. The normalized results can be compared from one experiment to another even when the experiments were performed using different fluorescent substrate concentrations and/or cell densities. Since changes in DCFH oxidation may be related to changes that are indicative of oxidative stress in cells, this assay can be useful to quantify radiation-induced oxidative stress and evaluate the efficacy of antioxidant agents in protection against radiation-induced oxidative stress.

journal_name

Radiat Res

journal_title

Radiation research

authors

Wan XS,Zhou Z,Ware JH,Kennedy AR

doi

10.1667/rr3299

subject

Has Abstract

pub_date

2005-02-01 00:00:00

pages

232-40

issue

2

eissn

0033-7587

issn

1938-5404

pii

RR3299

journal_volume

163

pub_type

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