Electroporation phenomena is a highly effective method in different fields of application, including cancer treatment [1]. This method is based on pulsed electric fields (PEF) and pore formation in cell membrane consequently causing the increase in cell membrane permeability [2]. The combination of electroporation with drugs, which is known as electrochemotherapy, is the highly effective method in cancer treatment [3]. Although the outcome depending on pulse parameters, and physical factors may vary. Hence the occurrence the permeabilization rate in vitro is quite frequently evaluated using fluorescent markers such as propidium iodide (PI), while the viability is checked after 24 hours or days using metabolic activity test or clonogenic assay. In this work, an alternative way for detection of the electroporation efficacy and drop of cell viability following PEF treatment is proposed. This method is based on oxidation of D-luciferin sodium salt utilized to assay the luciferase gene expression in stably transfected bioluminescent mice Sp2/0 myeloma cells.
A 0-3 kV, 60 A pulsed power generator (VGTU, Lithuania) was used in the study. Cells were treated in electroporation cuvette with 1 mm gap aluminum electrodes (Biorad, Hercules, USA). Pulses in the range of 1-2.5 kV/cm 0.1-100 µs were generated. Cell permeabilization efficiency was evaluated using propidium iodide (PI, Sigma-Aldrich) and flow cytometry, while viability was estimated using alamarBlue™ cell viability reagent (Thermo Fisher Scientific, USA) after 24 h. Alternatively, the Sp2/0 transfected cells (Luciferase-pcDNA3 plasmid, Adgene #18964) were treated by PEF and later transferred into the white 96-well plates. Subsequently, D-Luciferin (Promega, USA) was added to the cells, then analyzed concentration was 150 µg/ml. Synergy 2 microplate reader and Gen5 software (BioTek, USA) were used for SP2/0 cells luminescence evaluation.
It was shown that the bioluminescence signal was scaling in a dose dependent manner following the same tendency as permeabilization curves obtained using conventional toolkits. At the same time, it also provided a real-time feedback on cell viability since only viable cells luminesce. It was also shown that the extent of permeabilization and thus, the oxidation of D-luciferin is not the same for microsecond and sub-microsecond pulses, while bioluminescence still can be implemented as an effective method for analysis of electroporation phenomena in vitro.