Research and Development Department, Promega Corporation, Madison, WI 53711, USA.
Biotechniques. 2009 Sep;47(3):769-74. doi: 10.2144/000113220.
As a means of reliably measuring intracellular pH, we have precisely targeted 5(and 6-) carboxyseminaphthorhodafluor to cellular subcompartments. This was accomplished by combining the well-established pH-sensitive dye with a protein-based reporter system. When expressed in cells, the reporter protein is designed to covalently bind ligands composed of a functional group and a reactive linker. In order to make a pH-sensitive ligand, we chemically coupled the pH sensor to a reactive linker. Several ligands of differing linker lengths were made and tested for their pH responsiveness in vitro. The most responsive of these ligands was then evaluated for its efficacy in live cell labeling and its use as an intracellular pH sensor for ratiometric confocal microscopy. Here we show that we could target this pH sensor within mammalian cells exclusively to either the nucleus or cytoplasm. Exhibiting the versatility of this reporter technology, we were also able to specifically limit pH sensor labeling to within the trafficking pathway of integrins and directly measure pH of this environment. Results correspond well with previously published reports. Both the simplicity and flexibility of the technology used in this study make possible the development of diverse targeted microenvironmental sensors or other moieties of interest.
作为一种可靠测量细胞内 pH 值的手段，我们已经将 5（和 6-）羧基半花青染料精确定位到细胞亚区室。这是通过将成熟的 pH 敏感染料与基于蛋白质的报告系统相结合来实现的。当在细胞中表达时，报告蛋白被设计为共价结合由功能基团和反应性接头组成的配体。为了制作 pH 敏感配体，我们将 pH 传感器化学偶联到反应性接头。合成了几种具有不同接头长度的配体，并在体外测试它们的 pH 响应性。这些配体中最敏感的一种被评估其在活细胞标记中的功效及其作为比率荧光共焦显微镜的细胞内 pH 传感器的用途。在这里，我们证明我们可以将这种 pH 传感器专门靶向哺乳动物细胞的核或细胞质。展示了这种报告技术的多功能性，我们还能够将 pH 传感器标记特异性限制在整合素的运输途径内，并直接测量该环境的 pH 值。结果与之前发表的报告相符。本研究中使用的技术的简单性和灵活性使得开发各种靶向微环境传感器或其他感兴趣的部分成为可能。
