EVALUATION OF METHODS FOR MEASURING CELLULAR GLUTATHIONE CONTENT USING FLOW-CYTOMETRY
EVALUATION OF METHODS FOR MEASURING CELLULAR GLUTATHIONE CONTENT USING FLOW-CYTOMETRY
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DOI：
10.1002/cyto.990150411
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发表时间：
1994-04-01
期刊：
影响因子：
--
通讯作者：
CHOW, S
中科院分区：
文献类型：
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作者：
HEDLEY, DW;CHOW, S
评估了目前可用的细胞谷胱甘肽的流式细胞仪染色，检查了不同浓度、时间和温度条件下对人和啮齿动物细胞系的标记。使用耗尽谷胱甘肽(GSH)，同时对其他细胞巯基影响最小的程序，以估计线性和背景染色的程度。一氯二苯醚在啮齿动物细胞中对谷胱甘肽具有高度的特异性，但由于其对人谷胱甘肽S转移酶的亲和力较低，因此不能充分标记人类细胞。较高浓度的一氯双烷可以更完整地标记人细胞的GSH池，但由于没有GSH结合，背景荧光增加。类似物单溴双胺在非酶作用下与巯基结合，更容易与GSH反应，而不是与蛋白巯基反应，在控制染色浓度和孵育时间的情况下，由于背景染色，可对人类细胞进行重复性染色，约占总荧光的20%。在目前可用于测量人类细胞中GSR的染色方法中，单溴莫比胺是首选试剂。汞橙也更容易与GSH结合，而不是与蛋白质结合，具有一定的特异性，而且它还有一个额外的优势，即在488 nm处被激发。然而，汞橙染色的重现性不如一溴莫比胺，背景荧光较高。另外两种染料，邻苯二甲醛和氯甲基荧光素，也可以用来标记细胞GSH，但这两种染料的背景染色水平都高得令人无法接受。为了确定背景标记的程度，建议流式细胞仪GSH检测应常规包括已耗尽GSH的细胞样本。(C)1994年Wiley-Liss，Inc.
The currently available flow cytometric stains for cellular glutathione were evaluated, examining the labelling of both human and rodent cell lines under various conditions of concentration, time, and temperature. Procedures were used that depleted glutathione (GSH) while having a minimal effect on other cellular sulphydryls in order to estimate linearity and the extent of background staining. As previously reported, monochlorobimane was highly specific for GSH in rodent cells but failed to label human cells adequately because of its low affinity for human glutathione S-transferases. Higher concentrations of monochlorobimane achieved more complete labelling of the human cellular GSH pool but gave increased background fluorescence due to non-GSH binding. The analogue monobromobimane, which binds nonenzymatically to sulphydryls, reacted more readily with GSH than with protein sulphydryls and, provided that stain concentration and incubation time were controlled, gave reproducible staining of human cells with approximately 20% of total fluorescence due to background staining. Of the currently available stains for measuring GSR in human cells, monobromobimane is the agent of choice. Mercury orange also binds more readily to GSH than to protein, giving a degree of specificity, and it has the additional advantage of being excited at 488 nm. However, the reproducibility of staining with mercury orange was less consistent than that using monobromobimane, and a higher background fluorescence was seen. Two additional stains, o-phthaldialdehyde and chloromethyl fluorescein, could also be used to label cellular GSH, but both gave an unacceptably high level of background staining. It is recommended that flow cytometric GSH assays should routinely include a sample of cells that have been depleted of GSH in order to determine the extent of background labelling. (C) 1994 Wiley-Liss, Inc.