Webb Sarah E, Rogers Kelly L, Karplus Eric, Miller Andrew L
Biochemistry and Cell Biology Section and State Key Laboratory of Molecular Neuroscience, Division of Life Science, HKUST, Clear Water Bay, Kowloon, Hong Kong, PR China.
Methods Cell Biol. 2010;99:263-300. doi: 10.1016/B978-0-12-374841-6.00010-4.
In this chapter, we describe the practical aspects of measuring [Ca(2+)] transients that are generated in a particular cytoplasmic domain, or within a specific organelle or its periorganellar environment, using bioluminescent, genetically encoded and targeted Ca(2+) reporters, especially those based on apoaequorin. We also list examples of the organisms, tissues, and cells that have been transfected with apoaequorin or an apoaequorin-BRET complex, as well as of the organelles and subcellular domains that have been specifically targeted with these bioluminescent Ca(2+) reporters. In addition, we summarize the various techniques used to load the apoaequorin cofactor, coelenterazine, and its analogs into cells, tissues, and intact organisms, and we describe recent advances in the detection and imaging technologies that are currently being used to measure and visualize the luminescence generated by the aequorin-Ca(2+) reaction within these various cytoplasmic domains and subcellular compartments.
在本章中，我们描述了使用生物发光、基因编码且靶向的Ca(2+)报告分子，特别是基于脱辅基水母发光蛋白的报告分子，来测量在特定细胞质区域、特定细胞器或其细胞器周围环境中产生的[Ca(2+)]瞬变的实际操作。我们还列举了已转染脱辅基水母发光蛋白或脱辅基水母发光蛋白 - BRET复合物的生物体、组织和细胞的实例，以及这些生物发光Ca(2+)报告分子已特异性靶向的细胞器和亚细胞区域的实例。此外，我们总结了用于将脱辅基水母发光蛋白辅因子腔肠素及其类似物加载到细胞、组织和完整生物体中的各种技术，并描述了目前用于测量和可视化这些不同细胞质区域和亚细胞区室中水母发光蛋白 - Ca(2+)反应产生的发光的检测和成像技术的最新进展。
