Wang Guqi, Gong Yu, Burczynski Frank J, Hasinoff Brian B
Faculty of Pharmacy, University of Manitoba, Winnipeg, Manitoba, Canada.
Free Radic Res. 2008 May;42(5):435-41. doi: 10.1080/10715760802074462.
The oxidation of 2',7'-dichlorodihydrofluorescein (2',7'-dichlorofluorescin, DCFH) to a fluorescent product, 2',7'-dichlorofluorescein (DCF), is commonly used to quantitatively measure oxidative stress in cells using a fluorescence microplate reader. However, many cell lines tend to grow non-uniformly in the wells. This non-uniform distribution results in a high degree of variability in the fluorescence signal and decreases the precision of the method. Also, samples treated in large culture plates, dishes or flasks cannot be assayed directly in fluorescence microplate readers. This study reports an improved DCF assay method that lyses cells with DMSO/PBS (90% dimethyl sulphoxide/10% phosphate buffered saline). Oxidative stress was induced with either hydrogen peroxide or an hypoxia-reoxygenation treatment. Cell lysis with DMSO/PBS resulted in highly stable fluorescence signals in comparison to Triton X-100/PBS lysed cells. The precision of DCF fluorescence measurements of DMSO/PBS lysed cells was much better than for attached cells measured directly in 96-well plates. While DCF fluorescence in PBS was strongly quenched by albumin, no quenching occurred in DMSO/PBS. In conclusion this study describes a more convenient and accurate method for measuring cellular oxidative stress that also makes it possible to assay cells treated in large culture plates.
2',7'-二氯二氢荧光素（2',7'-二氯荧光素，DCFH）氧化为荧光产物2',7'-二氯荧光素（DCF），通常用于使用荧光酶标仪定量测量细胞中的氧化应激。然而，许多细胞系在孔中往往生长不均匀。这种不均匀分布导致荧光信号的高度变异性，并降低了该方法的精度。此外，在大培养板、培养皿或烧瓶中处理的样品不能直接在荧光酶标仪中进行检测。本研究报告了一种改进的DCF检测方法，该方法用DMSO/PBS（90%二甲基亚砜/10%磷酸盐缓冲盐水）裂解细胞。用过氧化氢或缺氧复氧处理诱导氧化应激。与用Triton X-100/PBS裂解的细胞相比，用DMSO/PBS裂解细胞产生了高度稳定的荧光信号。用DMSO/PBS裂解的细胞的DCF荧光测量精度比直接在96孔板中测量的贴壁细胞要好得多。虽然PBS中的DCF荧光被白蛋白强烈淬灭，但在DMSO/PBS中没有发生淬灭。总之，本研究描述了一种更方便、准确的测量细胞氧化应激的方法，该方法还使得检测在大培养板中处理的细胞成为可能。
