Decrock Elke, De Bock Marijke, De Baere Diego, Hoorelbeke Delphine, Wang Nan, Leybaert Luc
Physiology Group, Department of Basic Medical Sciences, Ghent University, De Pintelaan 185, Ghent, 9000, Belgium.
Methods Mol Biol. 2016;1437:155-69. doi: 10.1007/978-1-4939-3664-9_11.
Intercellular communication occurring via gap junction channels is considered a key mechanism for synchronizing physiological functions of cells and for the maintenance of tissue homeostasis. Gap junction channels are protein channels that are situated between neighboring cells and that provide a direct, yet selective route for the passage of small hydrophilic biomolecules and ions. Here, an electroporation method is described to load a localized area within an adherent cell monolayer with a gap junction-permeable fluorescent reporter dye. The technique results in a rapid and efficient labeling of a small patch of cells within the cell culture, without affecting cellular viability. Dynamic and quantitative information on gap junctional communication can subsequently be extracted by tracing the intercellular movement of the dye via time-lapse microscopy.
通过间隙连接通道进行的细胞间通讯被认为是使细胞生理功能同步以及维持组织稳态的关键机制。间隙连接通道是位于相邻细胞之间的蛋白质通道，为小的亲水性生物分子和离子的通过提供了一条直接但具有选择性的途径。在此，描述了一种电穿孔方法，用于用可透过间隙连接的荧光报告染料加载贴壁细胞单层内的局部区域。该技术可快速有效地标记细胞培养物中的一小片细胞，而不影响细胞活力。随后，通过延时显微镜追踪染料的细胞间移动，可以提取有关间隙连接通讯的动态和定量信息。
