## 3 Results
### Transfection Efficiency and Cell Viability Dependence on Plasmid Concentration
Figure 1: The dependence of cell electrotransfection efficiency (**A**) and total fluorescence (**B**) on the plasmid concentration after treatment with 1, 2 and 3 HV (1400 V/cm strength, 100 \(\upmu\)s duration) pulses at 1 Hz repetition frequency. The results represent the transfected cells and do not account for cell viability changes. The concentration of GFP coding plasmid was 10–600 \(\upmu\)g/mL. The point on each curve at 0 \(\upmu\)g/mL plasmid concentration represents the control point (electroporation (EP) alone). Error bars represent standard error of mean (SEM). The significance of differences was calculated using one-way ANOVA. 3 symbols *** represents differences between neighboring points after treatment with 1 HV, ##—after treatment with 2 HV, and $—after the treatment with 3 HV.  
Figure 2: The dependence of cell viability on plasmid concentration after treatment with 1, 2 and 3 HV (1400 V/cm, 100 \(\upmu\)s) pulses at 1 Hz repetition frequency. The concentration of GFP coding plasmid was 10–600 \(\upmu\)g/mL. The point on each curve at 0 \(\upmu\)g/mL plasmid concentration represents the control point (electroporation (EP) alone). Error bars represent standard error of mean (SEM). The significance of differences was calculated using one-way ANOVA. 3 symbols *** represents differences between neighboring points after treatment with 1 HV, ##—after treatment with 2 HV, and $—after the treatment with 3 HV. As we observed a large change in cell viability in dependence of the plasmid concentration, we proceeded to analyze the number of transfected cells in the population of all treated cells (Figure 3A). Additionally, we observed the total fluorescence in these transfected cells (Figure 3B). We can see that the differences between the electric pulse conditions and the general shape of the curve's changes in comparison to the ones measured on the surviving cells alone (see Figure 1 for comparison). After treatment with 1 HV pulse, the number of transfected cells increases with the increasing plasmid concentrations until 200 \(\upmu\)g/mL plasmid concentration. After that, the number of transfected cells reaches a plateau and does not significantly increase with the higher plasmid concentrations used. After the treatment with either two or three HV pulses, the plateau in the number of transfected cells is reached at 100 \(\upmu\)g/mL and stays unchanged with up to 400 \(\upmu\)g/mL plasmid concentrations used. However, when the plasmid concentration is increased up to 600 \(\upmu\)g/mL, the number of transfected cells starts to statistically significantly decrease. However, even though the number of transfected cells stays relatively similar over the plasmid concentration range of 200-400 \(\upmu\)g/mL, the total fluorescence of these cells does not. Irrespectively of the number of HV pulses used, the total fluorescence of the transfected cells increases up to 400 \(\upmu\)g/mL plasmid concentration. With one HV, the total fluorescence increases further when the plasmid concentration is raised to 600 \(\upmu\)g/mL. However, with two and three HV pulses, the increase of plasmid concentration to 600 \(\upmu\)g/mL causes the total fluorescence of the cell population to drop again, approaching similar level than the one seen with twice as low 300 \(\upmu\)g/mL plasmid concentration. Transfection Efficiency and Cell Viability Dependence on the Distance between Electroporated Cell Membrane and the Nearest Plasmid  
The electrotransfection of the cells is dependent on the electrophoretic forces as the diffusion of plasmid sized DNA molecules is low even in aqueous solutions [29]. Therefore, on the same electric field parameters, the distance between the plasmid DNA and the plasma membrane, which is determined by the plasmid concentration, will determine how many DNA molecules will be able to reach the membrane. In turn, this will impact the transfection efficiency.