Technical Reference #1687

1687.

In Situ Bipolar Electroporation for Localized Cell Loading with Reporter Dyes and Investigating Gap Junctional Coupling Elke De Vuyst; Marijke De Bock; Elke Decrock; Marijke Van Moorhem;* Christian Naus; y Cyriel Mabilde;* and Luc Leybaert*, Ghent University, Biophysical Journal, 94(1687), (2008)
Link To Paper

Abstract:
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.

Materials & Methods:
Confluent monolayer cultures were grown on glass-bottom microwells (MatTek Ashland MA) and were loaded with 5-CFDA (10 mM) in HBSSHEPES for 45 min at room temperature. Fluorescence within a single cell was photobleached by spot exposure to the 488-nm line of an argon laser and imaging (again at 488nmexcitation) was done with a custom-made video-rate confocal laser scanning microscope (37) with a 403 oil immersion objective (CFI Plan Fluor NA 1.4 Nikon Belux); recovery was then measured over a 5-min time period after photobleaching. In the experiments where supernatant of scraped cultures was used the supernatant was obtained 1 min after the scratch (conditions as described above) and acceptor cultures were exposed for 10 min before the start of the FRAP experiments.

Microscopic Technique
Confocal Microscopy, Laser Scanning

Cell Type(s)
ECV304