title: Fluorometric assay to compensate for non-viable cells during electroporation.
abstract: A fluorometric assay is described that allows adjustment for non-viable cells that result during electroporation. The technique, unlike others, relies on only one dye, requires a single instrument, and eliminates the need for a separate cell counting step. Murine melanoma (B16-F10) cells were electroporated using electric fields ranging from 400 to 2500 V/cm in the presence of SYTOX(®)-green. Compensation for the fluorescence resulting from non-viable cells was facilitated by a correction curve established by lysing a known number of cells in the presence of SYTOX(®)-green. In uncorrected data, an applied electric field of 2500 V/cm increased dye delivery but also reduced cell viability significantly. Compensating for the fluorescence of non-viable cells showed that changing the field strength to 800 V/cm or 2500 V/cm from 400 V/cm had only marginal effects on membrane pore formation. The fluorometric assay was used to compare electroporation in high conductivity (PBS) and a low conductivity medium (LC-PBS). Statistically significant increases of 10 to 30-fold were observed for cells electroporated at 400 V/cm and 800 V/cm in LC-PBS.
authors.affiliation: Department of Chemical and Biomedical Engineering, College of Engineering, University of South Florida, ENB 118, 4202 E. Fowler Ave, Tampa, FL, 33620, USA.
authors.forename: Alisha D
authors.initials: AD
authors.lastname: Peterson
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Jaroszeski
authors.forename: Vinay K
authors.initials: VK
authors.lastname: Gupta
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D002470:Cell Survival; D018274:Electroporation / Q000379:methods*; D005470:Fluorometry / Q000379:methods*; D051379:Mice