Nieminen A L, Gores G J, Bond J M, Imberti R, Herman B, Lemasters J J
Department of Cell Biology & Anatomy, School of Medicine, University of North Carolina, Chapel Hill 27599-7090.
Toxicol Appl Pharmacol. 1992 Aug;115(2):147-55. doi: 10.1016/0041-008x(92)90317-l.
A new assay using a multiwell fluorescence scanner was developed for screening cytotoxicity to cells cultured in 96-well microtiter plates. The assay is based on binding of propidium iodide to nuclei of cells whose plasma membranes have become permeable due to cell death. Fluorescence of propidium iodide measured with a multiwell fluorescence scanner increased in proportion to the number of permeabilized cells. After ATP depletion of hepatocytes and neonatal cardiac myocytes with metabolic inhibitors ("chemical hypoxia"), and exposure of Madine Darby canine kidney cells to the toxic chemical, HgCl2, propidium iodide fluorescence progressively increased. Increases of fluorescence were linearly proportional with release of lactate dehydrogenase into the culture medium. Employing this cytotoxicity screening assay, protection by various agents against lethal injury was evaluated in cultured hepatocytes during chemical hypoxia. Inhibitors of cysteine proteases (i.e., antipain, leupeptin, E-64), serine proteases (i.e., PMSF), and aspartic acid proteases (i.e., pepstatin A) did not protect against chemical hypoxia. In contrast, 1,10-phenanthroline, an inhibitor of metalloprotease, markedly protected against the onset of cell death during chemical hypoxia. Half-maximal protection after 60 min occurred at 0.5 microM. Phospholipase inhibitors, chlorpromazine (50 microM) and mepacrine (50 microM), also substantially retarded cell killing. U74006F, an inhibitor of lipid peroxidation, slowed cell killing to a lesser extent during chemical hypoxia and after oxidative stress with t-butyl hydroperoxide. Calciphor, a dimer of prostaglandin B1, did not protect against cell killing during chemical hypoxia or t-butyl hydroperoxide toxicity. In conclusion, this high capacity cytotoxicity assay for cells cultured in 96-well microtiter plates is suitable for rapid screening of potential cytoprotective agents in a variety of cell types.
开发了一种使用多孔荧光扫描仪的新检测方法，用于筛选对培养在96孔微量滴定板中的细胞的细胞毒性。该检测方法基于碘化丙啶与细胞膜因细胞死亡而变得通透的细胞的细胞核的结合。用多孔荧光扫描仪测量的碘化丙啶荧光与通透化细胞的数量成比例增加。在用代谢抑制剂（“化学性缺氧”）使肝细胞和新生心肌细胞的ATP耗竭，以及将Madine Darby犬肾细胞暴露于有毒化学物质HgCl2后，碘化丙啶荧光逐渐增加。荧光的增加与乳酸脱氢酶释放到培养基中的量呈线性比例。采用这种细胞毒性筛选检测方法，评估了在化学性缺氧期间各种试剂对培养的肝细胞致死性损伤的保护作用。半胱氨酸蛋白酶抑制剂（即抗蛋白酶、亮抑蛋白酶肽、E-64）、丝氨酸蛋白酶抑制剂（即苯甲基磺酰氟）和天冬氨酸蛋白酶抑制剂（即胃蛋白酶抑制剂A）对化学性缺氧没有保护作用。相比之下，金属蛋白酶抑制剂1,10-菲咯啉在化学性缺氧期间对细胞死亡的发生有显著保护作用。60分钟后半数最大保护作用出现在0.5微摩尔浓度。磷脂酶抑制剂氯丙嗪（50微摩尔）和米帕林（50微摩尔）也显著延缓了细胞死亡。脂质过氧化抑制剂U74006F在化学性缺氧期间和用过氧化叔丁基进行氧化应激后，使细胞死亡的速度减缓程度较小。前列腺素B1的二聚体钙泊三醇在化学性缺氧或过氧化叔丁基毒性期间对细胞死亡没有保护作用。总之，这种用于96孔微量滴定板中培养细胞的高容量细胞毒性检测方法适用于快速筛选多种细胞类型中潜在的细胞保护剂。
