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, G_q, G_i/0 or G_s as well. The serial agonist addition protocol has the potential to further strengthen the output of label-free analysis of GPCR activation.

中文翻译：

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通过使用系列激动剂暴露方案，提高无标记细胞测定的通量以研究G蛋白偶联受体的激活

在评估G蛋白偶联受体（GPCR）配体时，无标记的整体测定法（例如监测电极上细胞的阻抗）正变得越来越受欢迎。这些方法的优势在于能够以无创，高度自动化的方式提供集成的细胞响应，并具有取决于设备的时间分辨率，可低至几毫秒。随着要并行研究的样本数量的增加，可使用的时间分辨率降低了，但是一次性传感器阵列的成本可能会受到限制。受器官药理学方法的启发，我们研究了一种简单的系列激动剂加成分析方法，该方法在基于阻抗的细胞分析中规避了这些局限性。使用串行除了不断增加浓度的GPCR激动剂，同时连续监测样品的阻抗，我们能够在单层内源性表达组胺1受体（H1R）的U-373 MG细胞上建立内源性激动剂组胺的完整浓度-响应曲线。对于常规的平行激动剂添加方案和使用H1R拮抗剂（如美吡拉敏）的研究，该方法已得到验证。显示了系列激动剂加成测定法的适用性，也适用于其他GPCR，这些GPCR也已知通过经典的G蛋白途径之一G _q，G _{i / 0}或G _s进行信号传导。该系列添加激动剂 该协议有可能进一步加强GPCR激活的无标记分析输出。