Increasing the throughput of label-free cell assays to study the activation of G-protein-coupled receptors by using a serial agonist exposure protocol.
Increasing the throughput of label-free cell assays to study the activation of G-protein-coupled receptors by using a serial agonist exposure protocol.
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DOI：
10.1093/intbio/zyz010
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发表时间：
2019-03
期刊：
影响因子：
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通讯作者：
J. Stolwijk;M. Skiba;C. Kade;G. Bernhardt;A. Buschauer;H. Hübner;P. Gmeiner;J. Wegener
中科院分区：
文献类型：
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作者：
J. Stolwijk;M. Skiba;C. Kade;G. Bernhardt;A. Buschauer;H. Hübner;P. Gmeiner;J. Wegener
无标记的整体分析，例如监测细胞在电极上的阻抗，在g蛋白偶联受体（GPCR）配体的评估中越来越受欢迎。这些方法的优势在于提供集成的细胞响应，无创，高度自动化，并且与设备相关的时间分辨率低至几毫秒。然而，随着并行研究的样品数量的增加，可用的时间分辨率降低，一次性传感器阵列的成本可能会受到限制。受器官药理学协议的启发，我们研究了一种简单的系列激动剂添加试验，该试验绕过了基于阻抗的细胞试验的这些限制。通过连续增加GPCR激动剂浓度，同时持续监测样品的阻抗，我们能够在内源性表达组胺1受体（H1R）的U-373 MG细胞单层上建立内源性激动剂组胺的完整浓度-反应曲线。该方法在传统的平行激动剂添加方案和使用甲吡嗪等H1R拮抗剂的研究中得到了验证。系列激动剂添加试验的适用性也被证明适用于其他已知的通过典型g蛋白途径之一（Gq， Gi/0或Gs）发出信号的gpcr。系列激动剂添加方案有可能进一步加强GPCR激活的无标记分析的输出。
Label-free, holistic assays, monitoring, for example, the impedance of cells on electrodes, are gaining increasing popularity in the evaluation of G-protein-coupled receptor (GPCR) ligands. It is the strength of these approaches to provide the integrated cellular response non-invasively, highly automated and with a device-dependent time resolution down to several milliseconds. With an increasing number of samples to be studied in parallel, the available time resolution is, however, reduced and the cost for the disposable sensor arrays may become limiting. Inspired by protocols from organ pharmacology, we investigated a simple serial agonist addition assay that circumvents these limitations in impedance-based cellular assays. Using a serial addition of increasing concentrations of a GPCR agonist while continuously monitoring the sample's impedance, we were able to establish a full concentration-response curve for the endogenous agonist histamine on a single layer of U-373 MG cells endogenously expressing the histamine 1 receptor (H1R). This approach is validated with respect to conventional, parallel agonist addition protocols and studies using H1R antagonists such as mepyramine. Applicability of the serial agonist addition assay was shown for other GPCRs known for their signaling via one of the canonical G-protein pathways, Gq, Gi/0 or Gs as well. The serial agonist addition protocol has the potential to further strengthen the output of label-free analysis of GPCR activation.