Applying label-free dynamic mass redistribution technology to frame signaling of G protein-coupled receptors noninvasively in living cells
Applying label-free dynamic mass redistribution technology to frame signaling of G protein-coupled receptors noninvasively in living cells
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DOI：
10.1038/nprot.2011.386
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发表时间：
2011-11-01
期刊：
影响因子：
14.8
通讯作者：
Kostenis, Evi
中科院分区：
文献类型：
--
作者：
Schroeder, Ralf;Schmidt, Johannes;Kostenis, Evi
无标记动态质量再分布（DMR）是一种尖端的检测技术，能够实时检测活细胞中的整合细胞反应。它依赖于检测生物传感器涂覆的微孔板上的折射率变化，该变化源于传感器表面附近的总生物量的刺激诱导的变化。在这里，我们描述了一个详细的协议，应用DMR技术框架的G蛋白偶联受体的功能行为，传统上检查的基础上检测个别第二信使，如cAMP，Ca 2+或肌醇磷酸的终点测定。该方法可以很容易地适应不同的细胞背景（贴壁或悬浮），包括原代人细胞。实时记录可以在384孔微量滴定板中进行，并在2小时内完成，或者根据要解决的生物学问题，可以延长到几个小时。整个过程，包括细胞收获和DMR检测，需要1-2天。
Label-free dynamic mass redistribution (DMR) is a cutting-edge assay technology that enables real-time detection of integrated cellular responses in living cells. It relies on detection of refractive index alterations on biosensor-coated microplates that originate from stimulus-induced changes in the total biomass proximal to the sensor surface. Here we describe a detailed protocol to apply DMR technology to frame functional behavior of G protein-coupled receptors that are traditionally examined with end point assays on the basis of detection of individual second messengers, such as cAMP, Ca2+ or inositol phosphates. The method can be readily adapted across diverse cellular backgrounds (adherent or suspension), including primary human cells. Real-time recordings can be performed in 384-well microtiter plates and be completed in 2 h, or they can be extended to several hours depending on the biological question to be addressed. The entire procedure, including cell harvesting and DMR detection, takes 1-2 d.