Grecco Hernán E, Roda-Navarro Pedro, Fengler Sven, Bastiaens Philippe I H
Department of Systemic Cell Biology, Max Planck Institute for Molecular Physiology, Dortmund, Germany.
Methods Enzymol. 2011;500:37-58. doi: 10.1016/B978-0-12-385118-5.00003-7.
Signal transduction is mediated by posttranslational modifications (PTMs) of proteins in complex signaling networks. Quantifying PTM levels of multiple network components in response to a stimulus is therefore the key to understand how their concerted activities give rise to cellular function. We have shown that fluorescence lifetime imaging microscopy (FLIM) on cell arrays (CA-FLIM) provides a method to accurately quantify PTM levels of many proteins in situ. Herein, we describe the detailed protocol for CA-FLIM. Less than 2 days are needed from cell array preparation to data analysis, where the main limiting step is the 24h needed for transfection. After generating a single cell array containing 384 spots, it can be imaged and analyzed in less than 2h.
信号转导由复杂信号网络中蛋白质的翻译后修饰（PTM）介导。因此，量化多个网络组件对刺激的PTM水平是理解它们的协同活动如何产生细胞功能的关键。我们已经表明，细胞阵列上的荧光寿命成像显微镜（FLIM）（CA-FLIM）提供了一种在原位准确量化许多蛋白质PTM水平的方法。在此，我们描述了CA-FLIM的详细方案。从细胞阵列制备到数据分析所需时间不到2天，其中主要限制步骤是转染所需的24小时。生成包含384个点的单细胞阵列后，可在不到2小时内对其进行成像和分析。
