In situ bipolar electroporation for localized cell loading with reporter dyes and investigating gap junctional coupling.
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.
De Vuyst
Elke
E
Department of Physiology and Pathophysiology, Faculty of Medicine and Health Sciences, Ghent University, B-9000 Ghent, Belgium.
De Bock
Marijke
M
Decrock
Elke
E
Van Moorhem
Marijke
M
Naus
Christian
C
Mabilde
Cyriel
C
Leybaert
Luc
L
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D004396:Coloring Agents / Q000378:metabolism*; D018031:Connexin 43 / Q000378:metabolism; D017630:Connexins / Q000378:metabolism; D018274:Electroporation / Q000379:methods*; D036681:Fluorescence Recovery After Photobleaching; D017629:Gap Junctions / Q000378:metabolism*; D007150:Immunohistochemistry; D008970:Molecular Weight; D051381:Rats; D000097002:Gap Junction beta-1 Protein
D004396:Coloring Agents; D018031:Connexin 43; D017630:Connexins; D000097002:Gap Junction beta-1 Protein