Droste Miriam S, Biel Stefan S, Terstegen Lara, Wittern Klaus-Peter, Wenck Horst, Wepf Roger
Beiersdorf AG, Research Microscopy, Hamburg, Germany and FB Naturwissenschaftliche Technik, Hochschule für Angewandte Wissenschaften, Hamburg, Germany.
J Biomed Opt. 2005 Nov-Dec;10(6):064017. doi: 10.1117/1.2138011.
To maintain the intracellular concentration of ions and small molecules on osmotic challenges, nature has developed highly sophisticated transport systems for regulating water and ion content. An ideal measurement technique for volume changes of cells during osmotic challenges has to fulfil two requirements: it has to be osmotically inert, and it should allow online monitoring of cell volume changes. Here, a simple fluorescence microscopy-based approach is presented. Using fluorescein as a negative stain, it is possible to monitor cell volume changes without affecting the functionality of cell membranes and cell osmolarity. Measurement of Madine-Darby canine kidney (MDCK) cells after hypo- and hyperosmotic challenges reveals the main advantages of this approach: besides providing precise and reproducible quantitative data on reversible cell volume changes, the viability of the cells can be assessed directly by the appearance of stain in the cytoplasm. This becomes evident especially after hypo-osmotic challenge of glutaraldehyde-treated cells, which become leaky after fixation, followed by a massive volume change. This new approach represents a very sensitive measurement technique for cell volume changes resulting from water or ion flux, and thus seems to be an ideal tool for studying cell volume regulatory processes.
为了在渗透压挑战下维持细胞内离子和小分子的浓度，自然界已经发展出高度复杂的运输系统来调节水和离子含量。一种用于测量渗透压挑战期间细胞体积变化的理想技术必须满足两个要求：它必须是渗透压惰性的，并且应该允许在线监测细胞体积变化。在此，提出了一种基于简单荧光显微镜的方法。使用荧光素作为负染色剂，可以监测细胞体积变化而不影响细胞膜的功能和细胞渗透压。对低渗和高渗挑战后的Madine-Darby犬肾（MDCK）细胞进行测量揭示了这种方法的主要优点：除了提供关于可逆细胞体积变化的精确且可重复的定量数据外，还可以通过细胞质中染色剂的出现直接评估细胞的活力。这在戊二醛处理的细胞受到低渗挑战后尤为明显，这些细胞在固定后会变得渗漏，随后会发生大量体积变化。这种新方法代表了一种用于测量由水或离子通量引起的细胞体积变化的非常灵敏的技术，因此似乎是研究细胞体积调节过程的理想工具。
