Miniaturization of intracellular calcium functional assays to 1536-well plate format using a fluorometric imaging plate reader
Miniaturization of intracellular calcium functional assays to 1536-well plate format using a fluorometric imaging plate reader
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DOI：
10.1177/1087057104264038
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发表时间：
2004-08-01
影响因子：
--
通讯作者：
Strulovici, B
中科院分区：
文献类型：
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作者：
Hodder, P;Mull, R;Strulovici, B
活体哺乳动物细胞内钙反应瞬变的测量是一种流行的功能测定，用于鉴定具有药理学意义的受体或通道的激动剂和拮抗剂。近年来，基于荧光的检测技术和自动化技术的进步促进了该测定法适应 384 孔微孔板格式高通量筛选 (HTS) 测定法。然而，以 384 孔格式进行细胞内钙 HTS 测定所需的成本和时间对于大于 I x I 10(6) 孔的 HTS 活动可能会令人望而却步。由于这些原因，将细胞内钙功能测定小型化为 1536 孔微孔板形式很有吸引力，其中测定体积和板通量可以减少数倍。本文描述的研究重点是将细胞内钙测定小型化至 1536 孔板形式。这是通过修改荧光成像板读取器 (FLIPR) 的硬件和软件来实现的，以便能够将纳升测试化合物直接转移到 1536 孔测定板，并同时测量所有 1536 孔产生的钙响应。针对大鼠毒蕈碱乙酰胆碱受体亚型 I (rmAchR I) G 蛋白偶联受体 (GPCR) 的细胞内钙功能测定被小型化并在该改良仪器上进行。在测量已知毒蕈碱受体激动剂和拮抗剂活性的实验中，小型FLIPR测定给出了与384孔格式测定相当的EC50和IC50值以及排序效力。小型激动剂和拮抗剂测定的计算 Z' 因子分别为 0.56 +/- 0.21 和 0.53 +/- 0.22，比 384 孔测定中的相应值略高（Z'（激动剂）= 0.55 +/- 0.33）和更低（Z'（激动剂）= 0.70 +/- 0.18）。针对小型化毒蕈碱受体的模拟激动剂 HTS 活动能够识别所有掺有 rmAchR1 激动剂卡巴胆碱的孔。
The measurement of intracellular calcium response transients in living mammalian cells is a popular functional assay for identification of agonists and antagonists to receptors or channels of pharmacological interest. In recent years, advances in fluorescence-based detection techniques and automation technologies have facilitated the adaptation of this assay to 384-well microplate format high-throughput screening (HTS) assays. However, the cost and time required performing the intracellular calcium HTS assays in the 384-well format can be prohibitive for HTS campaigns of greater than I x I 10(6) wells. For these reasons, it is attractive to miniaturize intracellular calcium functional assays to the 1536-well microplate format, where assay volumes and plate throughput can be decreased by several fold. The focus of the research described in this article is the miniaturization of an intracellular calcium assay to 1536-well plate format. This was accomplished by modifying the hardware and software of a fluorometric imaging plate reader (FLIPR) to enable transfer of nanoliters of test compound directly to a 1536-well assay plate, and measure the resulting calcium response from all 1536 wells simultaneously. An intracellular calcium functional assay against the rat muscarinic acetylcholine receptor subtype I (rmAchR I) G-protein coupled receptor (GPCR) was miniaturized and executed on this modified instrument. In experiments measuring the activity of known muscarinic receptor agonists and antagonists, the miniaturized FLIPR assay gave EC50 and IC50 values and rank order potency comparable to the 384-well format assays. Calculated Z' factors for the miniaturized agonist and antagonist assays were, respectively, 0.56 +/- 0.21 and 0.53 +/- 0.22, which were slightly higher (Z'(agonist) = 0.55 +/- 0.33) and lower (Z'(agonist) = 0.70 +/- 0.18) than the corresponding values in the 384-well assays. A mock agonist HTS campaign against the muscarinic receptor in miniaturized format was able to identify all wells spiked with the rmAchR1 agonist carbachol.