Hedley D W, Chow S
Department of Pathology, Ontario Cancer Institute/Princess Margaret Hospital, Toronto, Canada.
Cytometry. 1994 Apr 1;15(4):349-58. doi: 10.1002/cyto.990150411.
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 GSH 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 labeling.
对目前可用的用于细胞内谷胱甘肽的流式细胞术染色剂进行了评估，研究了在浓度、时间和温度等各种条件下对人和啮齿动物细胞系的标记情况。采用了消耗谷胱甘肽（GSH）同时对其他细胞巯基影响最小的程序，以评估线性度和背景染色程度。如先前报道，一氯双硫腙对啮齿动物细胞中的GSH具有高度特异性，但由于其对人谷胱甘肽S-转移酶的亲和力低，未能充分标记人细胞。较高浓度的一氯双硫腙可使人细胞内GSH池的标记更完全，但由于非GSH结合导致背景荧光增加。与蛋白质巯基相比，非酶促结合巯基的类似物一溴双硫腙与GSH反应更迅速，并且只要控制染色剂浓度和孵育时间，就可对人细胞进行可重复染色，背景染色导致的总荧光约为20%。在目前可用的用于测量人细胞中GSH的染色剂中，一溴双硫腙是首选试剂。汞橙与GSH的结合也比与蛋白质的结合更迅速，具有一定程度的特异性，并且它还有在488nm激发的额外优势。然而，汞橙染色的可重复性不如一溴双硫腙，并且观察到更高的背景荧光。另外两种染色剂，邻苯二甲醛和氯甲基荧光素，也可用于标记细胞内GSH，但两者的背景染色水平都高得令人无法接受。建议流式细胞术GSH检测常规应包括一组已消耗GSH的细胞样本，以确定背景标记的程度。
