A one-day, dispense-only IP-one HTRF assay for high-throughput screening of Gαq protein-coupled receptors:: Towards cells as reagents
A one-day, dispense-only IP-one HTRF assay for high-throughput screening of Gαq protein-coupled receptors:: Towards cells as reagents
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DOI：
10.1089/adt.2007.108
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发表时间：
2008-02-01
影响因子：
1.8
通讯作者：
Bader, Benjamin
中科院分区：
文献类型：
--
作者：
Bergsdorf, Christian;Kropp-Goerkis, Carmen;Bader, Benjamin
与生物化学高通量筛选（HTS）测定相比，基于细胞的功能测定通常被认为更耗时且更复杂，这是因为在转移培养基和化合物时需要额外的努力来运行连续细胞培养物以及许多测定步骤。补偿预期的总通量降低的常见策略是实施高度自动化的细胞培养和筛选系统。然而，这种系统需要大量投资于尖端硬件和高度专业化的人员。在尝试建立替代方案以增加基于功能细胞的筛选中的通量时，我们结合了几种方法。通过使用（1）冷冻保存的细胞等分试样代替连续细胞培养物，（2）悬浮液中的细胞代替贴壁细胞，和（3）具有纳升等分试样的测试化合物的“准备筛选”测定板，开发了非常类似于标准生物化学、酶测定的测定程序，其仅包括几个分配步骤。使用稳定过表达G（α q）偶联受体的中国仓鼠卵巢细胞作为模型系统，通过在均匀时间分辨荧光（HTRF（R），CISbio International，巴尼奥尔-苏尔-策策，France）的帮助下检测细胞内D-肌肌醇1-磷酸来测量受体活化。最初在384孔贴壁细胞格式中建立，该测定成功地转移到1，536孔格式。测定质量足以以两种形式运行HTS活动，具有良好的Z '因子和优异的拮抗剂再现性。随后，对测定程序进行了优化，以使用悬浮细胞。评估了细胞培养基、平板类型、细胞数量和孵育时间的影响。最后，通过Schild图分析，将悬浮细胞试验应用于小分子拮抗剂的药理学表征。我们的数据不仅证明了IP-One HTRF测定（CISbio International）在高密度形式中用于HTS的应用，而且还证明了在一天功能性细胞测定中成功使用冷冻保存的和悬浮细胞。
Compared to biochemical high-throughput screening (HTS) assays, cell-based functional assays are generally thought to be more time consuming and complex because of additional efforts for running continuous cell cultures as well as the numerous assay steps when transferring media and compounds. A common strategy to compensate the anticipated reduction in overall throughput is to implement highly automated cell culture and screening systems. However, such systems require substantial investments in sophisticated hardware and highly specialized personnel. In trying to set up alternatives to increasing throughput in functional cell-based screening, we combined several approaches. By using (1) cryopreserved cell aliquots instead of continuous cell culture, (2) cells in suspension instead of adherent cells, and (3) "ready-to-screen" assay plates with nanoliter aliquots of test compounds, an assay procedure was developed that very much resembles a standard biochemical, enzymatic assay comprising only a few dispense steps. Chinese hamster ovary cells stably overexpressing a G(alpha q)-coupled receptor were used as a model system to measure receptor activation by detection of intracellular D-myo-inositol 1-phosphate with the help of homogeneous time-resolved fluorescence (HTRF (R), CISbio International, Bagnols-sur-Ceze, France). Initially established in 384-well adherent cell format, the assay was successfully transferred to 1,536-well format. The assay quality was sufficient to run HTS campaigns in both formats with good Z'-factors and excellent reproducibility of antagonists. Subsequently, the assay procedure was optimized for usage of suspension cells. The influences of cell culture media, plate type, cell number, and incubation time were assessed. Finally, the suspension cell assay was applied to pharmacological characterization of a small molecule antagonist by Schild plot analysis. Our data demonstrate not only the application of the IP-One HTRF assay (CISbio International) for HTS in a high-density format, but furthermore the successful use of cryopreserved and suspension cells in a one-day functional cell-based assay.