Cell lysis with dimethyl sulphoxide produces stable homogeneous solutions in the dichlorofluorescein oxidative stress assay
Cell lysis with dimethyl sulphoxide produces stable homogeneous solutions in the dichlorofluorescein oxidative stress assay
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DOI：
10.1080/10715760802074462
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发表时间：
2008-01
影响因子：
3.3
通讯作者：
Guqi Wang;Y. Gong;F. Burczynski;B. Hasinoff
中科院分区：
文献类型：
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作者：
Guqi Wang;Y. Gong;F. Burczynski;B. Hasinoff
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），通常用于荧光微孔板读取器定量测量细胞中的氧化应激。然而，许多细胞系在井中生长不均匀。这种不均匀分布导致荧光信号的高度可变性，降低了方法的精度。此外，在大培养皿、培养皿或烧瓶中处理的样品不能直接在荧光微孔板读取器中进行检测。本研究报告了一种改进的DCF检测方法，用DMSO/PBS（90%二甲基亚砜/10%磷酸盐缓冲盐水）裂解细胞。过氧化氢或低氧再氧化处理均可诱导氧化应激。与Triton X-100/PBS裂解细胞相比，DMSO/PBS裂解细胞产生高度稳定的荧光信号。DMSO/PBS裂解细胞的DCF荧光测量精度远高于直接在96孔板上测定的贴壁细胞。PBS中的DCF荧光被白蛋白强烈猝灭，而DMSO/PBS中未发生猝灭。总之，本研究描述了一种更方便和准确的测量细胞氧化应激的方法，也使测定在大培养板中处理的细胞成为可能。
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.