## 3 Results  
### Transfection Efficiency and Cell Viability Dependence on Plasmid Concentration  
The CHO-K1 cells were used in order to systematically determine the effect of different plasmid concentrations on electrotransfection efficiency. GFP coding plasmid concentrations from 10 to 600 ug/mL were used and the evaluation of transfection efficiency was performed by flow cytometry 24 h after electroporation. In our previous experiments we have determined that 1400 V/cm electric field strength yielded the most efficient electrotransfection of CHO-K1 cell line. Based on this, we used one, two, or three HV (1400 V/cm strength, 100 \(\upmu\)s duration) pulses at 1 Hz frequency for cell electroporation. The percentage of GFP positive cells measured 24 h after electroporation is presented in Figure 1A, and the total fluorescence in these cells is shown in Figure 1B. We can see that both the number of transfected cells (transfection efficiency) and the total fluorescence of the cell population statistically significantly increased with the increasing plasmid concentration. After treatment with 1 HV pulse, the number of transfected cells increases until 200 \(\upmu\)g/mL concentration, the rise slows down in the 200-400 \(\upmu\)g/mL range, but increases further after the plasmid concentration reaches 600 \(\upmu\)g/mL. After the application of two HV pulses, the number of transfected cells increases with the increasing plasmid concentration up to 300 \(\upmu\)g/mL, and then reaches the plateau, at least in the range of plasmid concentrations used. After the application of three HV pulses, the number of transfected cells increases up to 400 \(\upmu\)g/mL plasmid concentration, where it reaches 82%, which is the highest number of transfected cells achieved in all used conditions. However, the increase in plasmid concentration to 600 \(\upmu\)g/mL when three HV pulses were used caused a statistically significant drop in transfection efficiency. The total fluorescence of the cells follows the same trend as the number of transfected cells, although no decrease in total cell fluorescence between 400-600 \(\upmu\)g/mL plasmid concentrations after the treatment with three HV pulses is observed (see Figure 1B). It can also be seen that both the transfection efficiency and the total fluorescence of the cells is higher when a higher number of electric pulses is used. The most interesting observation is the observed decrease in transfection efficiency after treatment with three HV pulses when the plasmid concentration changes from 400 \(\upmu\)g/mL to 600 \(\upmu\)g/mL. This discrepancy has prompted us to evaluate the cell viability after electrotransfection. The results of these experiments are shown in Figure 2. With 1 HV pulse, the cell viability statistically significantly decreases in comparison to the control (0 \(\upmu\)g/mL plasmid concentration) at 200 \(\upmu\)g/mL plasmid concentration, and no statistically significant differences were detected between cell viability after electrotransfection with plasmid concentrations higher than 200 \(\upmu\)g/mL. A similar trend is observedafter electroporation with 2 HV pulses. In these conditions, cell viability remains stable in the plasmid concentration range of 200-400 \(\upmu\)g/mL but decreases further when a 600 \(\upmu\)g/mL concentration is used. After the treatment with three HV pulses, the cell viability steadily decreases over the whole range of plasmid concentrations used, although statistical significance is not always observed between the closest neighboring points. Overall, cell viability decrease is higher after the application of three HV pulses than after the application of one or two HV pulses. The highest loss of cell viability is observed with three HV pulses and 600 \(\upmu\)g/mL plasmid concentration, where it falls to \(\sim\)25%.