Miniaturization of a mammalian cell-based assay: Luciferase reporter gene readout in a 3 microliter 1536-well plate
Miniaturization of a mammalian cell-based assay: Luciferase reporter gene readout in a 3 microliter 1536-well plate
复制标题
DOI：
10.1177/108705719900400307
复制
发表时间：
1999-06-01
影响因子：
--
通讯作者：
Oldenburg, KR
中科院分区：
文献类型：
--
作者：
Maffia, AM;Kariv, I;Oldenburg, KR
组合化学和基因组学领域的共同努力显着增加了可用于筛选的化合物和治疗靶点的数量。在不久的将来，化合物的数量将达到百万级，并为药物发现提供巨大的化学多样性。然而，这种化学多样性库给任何筛选工作带来了下游障碍。正确检查如此数量的化合物会大大增加投资，无论是花费的美元数量还是消耗的有限试剂的数量。传统的高温超导技术（例如使用 96 孔微量滴定板）为更快的处理速度铺平了道路，但很快就被筛选需求所淹没。此类测定的小型化将允许更大的通量，同时降低成本。迄今为止，小型化工作已最成功地应用于基于细菌和可溶性蛋白质的测定。人们对在不破坏细胞膜和/或激活应激反应的情况下输送微量哺乳动物细胞的能力提出了疑问。已经开发出一种检测方法，其中人类 T 细胞筛选已适应 1536 孔板格式。通过使用荧光素酶报告基因系统，表明可以在 3μl 中成功地进行基于哺乳动物细胞的测定，并鉴定出感兴趣靶标的有效抑制剂。
The combined efforts of the fields of combinatorial chemistry and genomics have significantly increased the number of compounds and therapeutic targets available for screening. The number of compounds will reach into the million range in the near future and provide vast chemical diversity for drug discovery. However, this reservoir of chemical diversity creates downstream hurdles for any screening effort. Properly examining this number of compounds increases investments dramatically, both in the number of dollars spent and amount of limited reagents depleted. Traditional HTS techniques, such as the use of 96-well microtiter plates, have paved the way for faster processing speeds, but are being rapidly overwhelmed by screening demands. Miniaturization of such assays will allow for greater throughput, while concurrently reducing cost. To date, miniaturization efforts have been most successfully applied to bacterial and soluble protein based assays. Questions about the ability to deliver microquantities of mammalian cells without disruption of the cell membrane and/or activation of stress responses have been raised. An assay has been developed in which a human T-cell screen has been adapted to a 1536-well plate format. Through the use of a luciferase reporter gene system, it is shown that a mammalian cell-based assay may be successfully performed in 3 mu l and potent inhibitors of the target of interest identified.