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1.发明申请Continuous Assay Method for Measuring Receptor- and Ion Channel-Mediated Calcium Response and Subsequent Gene Expression 审中-公开用于测量受体和离子通道介导的钙响应和随后的基因表达的连续测定方法公开(公告)号：US20100159449A1
公开(公告)日：2010-06-24
申请号：US12302289
申请日：2007-05-28
CPC分类号： G01N33/6872 , C12Q1/66 , G01N33/5023 , G01N33/84 , G01N2333/726
摘要： To measure a receptor-mediated increase in intracellular calcium concentration, use is made of a calcium-responding luminescent protein localized in the cytoplasm or mitochondria and a reporter gene having a promoter responding to the second messenger in cells and Renilla-origin luciferase attached to the downstream thereof. By using a coelenterazine derivative, which can exist in the cells in a stable state over a long time, as the substrate of these proteins, a transient increase in the intracellular calcium concentration observed within several seconds is measured by using the calcium-responding luminescent protein. Subsequently, the cells are allowed to survive and cultured for several hours as such without adding a fresh substrate. During this period, an increase/decrease in the gene expression is continuously monitored by the reporter assay method and it is measured and evaluated, if necessary, whether or not the inhibition of the activity is caused by cytotoxicity.
摘要翻译： 为了测量受体介导的细胞内钙浓度的增加，使用定位在细胞质或线粒体中的钙响应的发光蛋白质，以及具有响应于细胞中的第二信使的启动子的报告基因和附着于细胞内的海肾起源的荧光素酶 下游。 通过使用长时间以稳定状态存在于细胞中的肠腔菌素衍生物作为这些蛋白质的底物，通过使用钙响应型发光蛋白质来测定数秒内观察到的细胞内钙浓度的瞬时增加 。 随后，允许细胞存活并培养数小时，而不添加新鲜的底物。 在此期间，通过报道分析方法不断监测基因表达的增加/减少，如果需要，可以测量和评估活性的抑制是否由细胞毒性引起。
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