Aeschbacher M, Reinhardt C A, Zbinden G
Institute of Toxicology, Federal Institute of Technology, Schwerzenbach, Switzerland.
Cell Biol Toxicol. 1986 Jun;2(2):247-55. doi: 10.1007/BF00122693.
A reliable and rapid test to detect cytotoxic chemicals which affect cell membranes is described. Fluorescein diacetate freely penetrates intact cells where it is hydrolyzed to its fluorochrome, fluorescein, which is retained in the cell due to its polarity. On the other hand, ethidium bromide is known to be excluded from the intact cell, staining only nucleic acids of membrane-damaged cells. The combination of both fluorochromes results in counter-staining: intact cells fluoresce green (cytoplasm) and membrane-damaged cells fluoresce red (nucleus and RNA). Rat thymocytes freshly isolated without enzyme treatment were incubated simultaneously with test substance and dye solution fluorescein diacetate and ethidium bromide. A two-parameter analysis was performed on a flow cytometer with an on-line computer. Concentration-dependent effects of various detergents and solvents were quantified by measuring the amount of dye retention, i.e., the decrease or increase in fluorescein--fluorescence (peak shift), and the decrease in dye exclusion (increase in ethidium bromide-staining) relative to the untreated control. The assay can be used for rapid monitoring of chemical insults to cell membranes which precede the decrease of the viability measured by pure dye exclusion techniques.
本文描述了一种可靠且快速的检测影响细胞膜的细胞毒性化学物质的方法。二乙酸荧光素可自由穿透完整细胞，在细胞内水解为荧光染料荧光素，由于其极性，荧光素会保留在细胞内。另一方面，已知溴化乙锭会被完整细胞排斥，仅对膜受损细胞的核酸进行染色。两种荧光染料的组合会产生复染效果：完整细胞发出绿色荧光（细胞质），膜受损细胞发出红色荧光（细胞核和RNA）。将未经酶处理新鲜分离的大鼠胸腺细胞与受试物质以及荧光素二乙酸酯和溴化乙锭的染料溶液同时孵育。使用在线计算机的流式细胞仪进行双参数分析。通过测量染料保留量，即荧光素荧光的减少或增加（峰位移）以及相对于未处理对照染料排斥的减少（溴化乙锭染色增加），对各种洗涤剂和溶剂的浓度依赖性效应进行定量。该检测方法可用于快速监测在通过纯染料排斥技术测量的活力降低之前对细胞膜的化学损伤。
