用于测量表达重组S1P受体亚型的细胞内Ca²⁺动员的高通量检测方法。
High-throughput assays to measure intracellular Ca²⁺ mobilization in cells that express recombinant S1P receptor subtypes.
作者信息
Valentine William J, Tigyi Gabor
机构信息
Department of Physiology, The University of Tennessee Health Science Center, Memphis, TN, USA.
出版信息
Methods Mol Biol. 2012;874:77-87. doi: 10.1007/978-1-61779-800-9_7.
Abstract
Intracellular Ca(2+) mobilization is a useful readout to screen for agonists or antagonists of G-protein -coupled receptors (GPCRs). Here, we describe methods to conduct high-throughput screening of stably or transiently transfected HTC4 cells expressing the individual S1P1-5 receptor subtypes. The cells are grown in 96-well plates and loaded with the cell permeable fluorescent Ca(2+) indicator dye Fura-2-AM. Changes in intracellular Ca(2+) levels in response to S1P or test compounds are detected using a FlexStation II scanning fluorometer with integrated fluidics transfer capabilities.
摘要
细胞内钙离子动员是筛选G蛋白偶联受体（GPCR）激动剂或拮抗剂的一种有用的检测方法。在此，我们描述了对稳定或瞬时转染表达单个S1P1 - 5受体亚型的HTC4细胞进行高通量筛选的方法。细胞在96孔板中培养，并加载可透过细胞的荧光钙指示剂染料Fura - 2 - AM。使用具有集成流体转移功能的FlexStation II扫描荧光计检测对S1P或测试化合物作出反应时细胞内钙离子水平的变化。
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