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.