De Vuyst Elke, De Bock Marijke, Decrock Elke, Van Moorhem Marijke, Naus Christian, Mabilde Cyriel, Leybaert Luc
Department of Physiology and Pathophysiology, Faculty of Medicine and Health Sciences, Ghent University, B-9000 Ghent, Belgium.
Biophys J. 2008 Jan 15;94(2):469-79. doi: 10.1529/biophysj.107.109470. Epub 2007 Sep 14.
Electroporation is generally used to transfect cells in suspension, but the technique can also be applied to load a defined zone of adherent cells with substances that normally do not permeate the plasma membrane. In this case a pulsed high-frequency oscillating electric field is applied over a small two-wire electrode positioned close to the cells. We compared unipolar with bipolar electroporation pulse protocols and found that the latter were ideally suited to efficiently load a narrow longitudinal strip of cells in monolayer cultures. We further explored this property to determine whether electroporation loading was useful to investigate the extent of dye spread between cells coupled by gap junctions, using wild-type and stably transfected C6 glioma cells expressing connexin 32 or 43. Our investigations show that the spatial spread of electroporation-loaded 6-carboxyfluorescein, as quantified by the standard deviation of Gaussian dye spread or the spatial constant of exponential dye spread, was a reliable approach to investigate the degree of cell-cell coupling. The spread of reporter dye between coupled cells was significantly larger with electroporation loading than with scrape loading, a widely used method for dye-coupling studies. We conclude that electroporation loading and dye transfer is a robust technique to investigate gap-junctional coupling that combines minimal cell damage with accurate probing of the degree of cell-cell communication.
电穿孔通常用于转染悬浮细胞，但该技术也可用于将通常不能透过质膜的物质加载到贴壁细胞的特定区域。在这种情况下，将脉冲高频振荡电场施加在靠近细胞的小型两线电极上。我们比较了单极和双极电穿孔脉冲方案，发现后者非常适合有效地加载单层培养物中狭窄的纵向细胞条带。我们进一步探索了这一特性，以确定电穿孔加载是否有助于研究通过缝隙连接耦合的细胞之间染料扩散的程度，我们使用了表达连接蛋白32或43的野生型和稳定转染的C6胶质瘤细胞。我们的研究表明，通过高斯染料扩散的标准差或指数染料扩散的空间常数来量化，电穿孔加载的6-羧基荧光素的空间扩散是研究细胞间耦合程度的可靠方法。与刮擦加载（一种广泛用于染料耦合研究的方法）相比，电穿孔加载时报告染料在耦合细胞之间的扩散明显更大。我们得出结论，电穿孔加载和染料转移是一种强大的技术，可用于研究缝隙连接耦合，该技术将最小的细胞损伤与对细胞间通讯程度的精确探测相结合。
