## 3 Results
The number of PI-positive cells obtained 15 min after pulse application revealed that membrane pores had already resealed within this period for the full range of CaCl\({}_{2}\) concentrations tested (Figure 5B). Even though the integrity of the cell membranes was restored, the results (Figure 3B,C) proposed that the cell viability was strongly affected within 24 h after calcium electroporation, which suggested that cell death mechanisms--independent from resealing of pores--were triggered as a response to the treatment with Ca\({}^{2+}\). Figure 4: The dependence of cell death on the applied electric field at different CaCl\({}_{2}\) concentrations. Cells were treated using one square pulse of 1200 V/cm for 250 \(\upmu\)s (**A**) or 1400 V/cm for 100 \(\upmu\)s (**B**). Results were evaluated 24 h after electroporation. Error bars represent the standard error of the mean (SEM) of \(n=9\) experimental replicates. In case standard errors were lower than actual data points, error bars are not visible. The statistical significance between the control (0 mM) and the 0.1, 0.01, 0.25, 0.5, and 1 mM CaCl\({}_{2}\) concentrations is denoted by *** (cell death induced by calcium electroporation) and *** (cell death induced by pDNA). *** and *** represents the \(p<0.001\). We also evaluated the importance of different plasmid DNA concentrations for the decrease in the efficiency of DNA electrotransfer induced by Ca\({}^{2+}\). As already described, electroporation of 1200 V/cm for 250 us resulted in a significantly higher electrotransfer efficiency, and therefore the experiments were performed using these electric field parameters (Figure 6). The increase in pDNA concentration resulted in a significant increase in the transfection efficiency (at 0 mM CaCl\({}_{2}\)) (Figure 6A). A similar tendency was observed when the CaCl\({}_{2}\) concentration was increased to 0.01 mM and 0.1 mM. Conversely, when the CaCl\({}_{2}\) concentration was further increased to 0.25 mM, an increase in the pDNA concentration did not further result in an increase in the transfection efficiency. The administration of a 0.01 mM CaCl\({}_{2}\) concentration to the electroporation medium resulted in an increase in the transfection efficiency from 24.3% (50 ug/mL) to 61.1% (200 ug/mL), whereas only an insignificant increase in the transfection efficiency was observed when the electroporation was performed at the 0.25 mM concentration of CaCl\({}_{2}\). Again, these results implied that 0.25 mM CaCl\({}_{2}\) is a threshold value in the process of pDNA electrotransfection and is important to the changes in cell viability. The successive increase in the CaCl\({}_{2}\) concentration supported this hypothesis, and no further increase in the transfection efficiency was observed when the CaCl\({}_{2}\) concentration in the medium was \(\geq\)0.5 mM. As expected, the evaluation of the total fluorescence intensity at different pDNA concentrations revealed an increase in fluorescence in accordance with the increasing plasmid concentration (Figure 6B). In addition, in agreement with previous results, the increase in the CaCl\({}_{2}\) concentration resulted in a gradual decrease in the total fluorescence for all pDNA concentrations used. The evaluation of the cell death (Figure 6C) indicated that the cell death occurring during Ca\({}^{2+}\) electroporation was dependent on the pDNA concentration. Nevertheless, the results also confirmed previous observations: when the CaCl\({}_{2}\) concentration exceeded 0.25 mM, the combination of Ca\({}^{2+}\) and pDNA significantly affected the cell viability. In this case, the viability decreased from 66.4% (electroporation alone) to 44.5, 28.8, 25.5, and 23.8% when plasmid concentrations of 50, 100, 150, and 200 ug/mL were used, respectively. However, only a slight decrease in the cell viability was observed when lower CaCl\({}_{2}\) (0.01 and 0.1 mM) concentrations and various concentrations of pDNA were used.