The Systematic Study of the Electroporation and Electrofusion of B16-F1 and CHO Cells in Isotonic and Hypotonic Buffer
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The fusogenic state of the cell membrane can be induced by external electric field. When two fusogenic membranes are in close contact, cell fusion takes place. An appropriate hypotonic treatment of cells before the application of electric pulses significantly improves electrofusion efficiency. How hypotonic treatment improves electrofusion is still not known in detail. Our results indicate that at given induced transmembrane potential electroporation was not affected by buffer osmolarity. In contrast to electroporation, cells’ response to hypotonic treatment significantly affects their electrofusion. High fusion yield was observed when B16-F1 cells were used; this cell line in hypotonic buffer resulted in 41 ± 9 % yield, while in isotonic buffer 32 ± 11 % yield was observed. Based on our knowledge, these fusion yields determined in situ by dual-color fluorescence microscopy are among the highest in electrofusion research field. The use of hypotonic buffer was more crucial for electrofusion of CHO cells; the fusion yield increased from below 1 % in isotonic buffer to 10 ± 4 % in hypotonic buffer. Since the same degree of cell permeabilization was achieved in both buffers, these results indicate that hypotonic treatment significantly improves fusion yield. The effect could be attributed to improved physical contact of cell membranes or to enhanced fusogenic state of the cell membrane itself.
KeywordsElectroporation Electrofusion Isotonic buffer Hypotonic buffer B16-F1 CHO Fluorescence microscopy
This research was supported by the Slovenian Research Agency under research programs P2-0249 and MRIC UL IP-0510. Research was conducted in the scope of the EBAM European Associated Laboratory. The authors thank Barbara Mali for her help with statistics.
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