Cell electrofusion visualized with fluorescence microscopy.
Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves application of short high-voltage electric pulses to cells that are in close contact. Application of short, high-voltage electric pulses causes destabilization of cell plasma membranes. Destabilized membranes are more permeable for different molecules and also prone to fusion with any neighboring destabilized membranes. Electrofusion is thus a convenient method to achieve a non-specific fusion of very different cells in vitro. In order to obtain fusion, cell membranes, destabilized by electric field, must be in a close contact to allow merging of their lipid bilayers and consequently their cytoplasm. In this video, we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method. In this method, cells are allowed to attach only slightly to the surface of the well, so that medium can be exchanged and cells still preserve their spherical shape. Fusion visualization is assessed by pre-labeling of the cytoplasm of cells with different fluorescent cell tracker dyes; half of the cells are labeled with orange CMRA and the other half with green CMFDA. Fusion yield is determined as the number of dually fluorescent cells divided with the number of all cells multiplied by two.
Trontelj
Katja
K
Faculty of Electrical Engineering, University of Ljubljana, Slovenia.
Usaj
Marko
M
Miklavcic
Damijan
D
D000818:Animals; D002459:Cell Fusion / Q000379:methods*; D004558:Electric Stimulation / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry; D008546:Melanoma, Experimental / Q000473:pathology; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*
D005456:Fluorescent Dyes