Villalobos Carlos, Alonso María Teresa, García-Sancho Javier
Instituto de Biología y Genética Molecular, Universidad de Valladolid and Consejo Superior de Investigaciones Científicas, Valladolid, Spain.
Methods Mol Biol. 2009;574:203-14. doi: 10.1007/978-1-60327-321-3_17.
Ca(2+) oscillations inside intracellular organelles are important for regulation of functions such as gene expression at the nucleus, respiration at mitochondria or protein processing at the endoplasmic reticulum. Targeted aequorins are excellent calcium probes for subcellular analysis, but single-cell imaging has proven difficult because of low light yield. Here we describe a procedure that combines virus-based expression of targeted aequorins with photon-counting imaging. This methodology allows real-time resolution of changes of cytosolic, mitochondrial or nuclear Ca(2+) signals at the single-cell level.
细胞内细胞器中的钙离子振荡对于调节诸如细胞核中的基因表达、线粒体中的呼吸作用或内质网中的蛋白质加工等功能非常重要。靶向水母发光蛋白是用于亚细胞分析的优秀钙探针，但由于光产量低，单细胞成像已被证明很困难。在这里，我们描述了一种将基于病毒的靶向水母发光蛋白表达与光子计数成像相结合的方法。这种方法能够在单细胞水平实时解析胞质、线粒体或细胞核钙离子信号的变化。
