title: Electrofusion of B16-F1 and CHO cells: the comparison of the pulse first and contact first protocols.
abstract: High voltage electric pulses induce permeabilisation (i.e. electroporation) of cell membranes. Electric pulses also induce fusion of cells which are in contact. Contacts between cells can be established before electroporation, in so-called contact first or after electroporation in pulse first protocol. The lowest fusion yield was obtained by pulse first protocol (0.8%±0.3%) and it was only detected by phase contrast microscopy. Higher fusion yield detected by fluorescence microscopy was obtained by contact first protocol. The highest fusion yield (15%) was obtained by modified adherence method whereas fusion yield obtained by dielectrophoresis was lower (4%). The results are in agreement with current understanding of electrofusion process and with existing electrochemical models. Our data indicate that probability of stalk formation leading to fusion pores and cytoplasmic mixing is higher in contact first protocol where cells in contact are exposed to electric pulses. Another contribution of present study is the comparison of two detection methods. Although fusion yield can be more precisely determined with fluorescence microscopy we should note that by using this detection method single coloured fused cells cannot be detected. Therefore low fusion yields are more reliably detected by phase contrast microscopy.
authors.affiliation: Faculty of Electrical Engineering, University of Ljubljana, Trzaska 25, SI-1000 Ljubljana, Slovenia.
authors.forename: Marko
authors.initials: M
authors.lastname: Usaj
authors.forename: Karel
authors.initials: K
authors.lastname: Flisar
authors.forename: Damijan
authors.initials: D
authors.lastname: Miklavcic
authors.forename: Masa
authors.initials: M
authors.lastname: Kanduser
mesh_terms: D000818:Animals; D016466:CHO Cells; D002448:Cell Adhesion; D002459:Cell Fusion / Q000379:methods*; D002498:Centrifugation; D006224:Cricetinae; D003412:Cricetulus; D004560:Electricity; D004586:Electrophoresis; D008546:Melanoma, Experimental / Q000473:pathology*; D051379:Mice