Standardization of a fluorometric assay for measuring oxidative stress in irradiated cells
Standardization of a fluorometric assay for measuring oxidative stress in irradiated cells
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DOI：
10.1667/rr3299
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发表时间：
2005-02-01
影响因子：
3.4
通讯作者：
Kennedy, AR
中科院分区：
文献类型：
--
作者：
Wan, XS;Zhou, ZZ;Kennedy, AR
本研究旨在标准化二氯荧光素(DCF)检测MCF-10细胞辐射诱导底物二氯荧光素(DCFH)氧化的方法。该方法具有很高的灵敏度，能够以线性的剂量-反应曲线检测低剂量的伽马射线照射后细胞中DCFH氧化的增加。然而，剂量-反应曲线的斜率在不同的实验之间有很大的不同，并且受到荧光底物浓度和细胞密度的影响。为了使分析具有重现性，以便比较不同实验获得的结果，建立了一系列换算因子和方程，以归一化这些变量的数据。结果表明，本研究中标准化的DCF测定具有高度的重复性和可接受的测定精密度。即使当实验使用不同的荧光底物浓度和/或细胞密度时，归一化结果也可以从一个实验到另一个实验进行比较。由于DCFH氧化的变化可能与细胞内氧化应激的变化有关，该方法可用于定量辐射诱导的氧化应激，并评估抗氧化剂对辐射诱导的氧化应激的保护作用。(C)2005年，由辐射研究学会提供。
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. (C) 2005 by Radiation Research Society.