## 3 Results  
The first part of the research was designed to evaluate whether low (0-1 mM) concentrations of CaCl\({}_{2}\) in the electroporation medium altered the efficacy of electrotransfection. The experiments were performed using two sets of pre-optimized electrical parameters and an optimal (high GET and high cell viability) pDNA concentration. The results, which are presented in Figure 2A, indicated that at 0 mM CaCl\({}_{2}\), a higher DNA transfection efficiency was obtained at a lower electric field strength (1200 V/cm) and a longer pulse duration (250 \(\upmu\)s) as compared with a higher electric field strength (1400 V/cm) and a shorter (100 \(\upmu\)s) pulse [11,22]. Interestingly, the pre-administration of CaCl\({}_{2}\) to the electroporation medium resulted in an inversely proportional dependence between the increment in the CaCl\({}_{2}\) concentration and the transfection efficiency (Figure 2A) as well as between the CaCl\({}_{2}\) concentration and the total fluorescence intensity (Figure 2B). Even low CaCl\({}_{2}\) concentrations (0.1 and 0.25 mM) in the electroporation medium induced a gradually decreasing GET and total fluorescence intensity in both electroporation protocols. The first relevant effect (\(p<0.001\)) was observed when the CaCl\({}_{2}\) concentration was increased to 0.25 mM. In this case, the transfection efficiency decreased from 53.4% (0 mM) to 22.8% (0.25 mM) when an electric field of 1200 V/cm was applied and from 36.4% (0 mM) to 14.03% (0.25 mM) when a single pulse of 1400 V/cm was used. Further increases in CaCl\({}_{2}\) to 0.5 and 1 mM resulted in a drastic decrease (\(p<0.001\)) in the number of GFP-positive cells as well as in the total fluorescence intensity. Since the results suggested that GET was significantly affected by CaCl\({}_{2}\), in the next part of the study we evaluated the influence of CaCl\({}_{2}\) on the cell viability. The data showed that calcium electroporation alone significantly reduced the cell viability when the CaCl\({}_{2}\) concentration exceeded 0.5 mM and an electric field of 1200 V/cm was used (Figure 3A). Even though such a significant decrease was not observed in the application of a 1400 V/cm strength pulse (Figure 3B), the simultaneous electrotransfer of pDNA and calcium revealed that a common additional cytotoxic effect occurred in a concentration-dependent manner for both electroporation protocols. Although very low concentrations of CaCl\({}_{2}\) (0.01-0.1 mM) in the electroporation medium induced only a slight decrease in the cell viability, the highest decrease was observed at the 0.25 mM concentration of CaCl\({}_{2}\): the viability decreased from 66.4 to 28.8% (1200 V/cm) and from 79.7 to 22.2% (1400 V/cm). Figure 2: Dependence of pDNA electrotransfection efficiency (**A**) and total fluorescence intensity (**B**) on the strength of the applied electric field at different CaCl\({}_{2}\) concentrations. Cells were treated using one square pulse of 1200 V/cm for 250 \(\upmu\)s or 1400 V/cm for 100 \(\upmu\)s. 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 0.01, 0.1, 0.25, 0.5, and 1 mM CaCl\({}_{2}\) concentrations is denoted by *** (1200 V/cm) and #, ##, and *** (1400V/cm). # represents \(p<0.05\), ##–\(p<0.01\), and *** or *** represents the \(p<0.001\). In order to check whether the observed reduction in GET was the result of cell death, we normalized the transfection efficiency to the number of viable cells (Figure 3C). The obtained results revealed that when the CaCl\({}_{2}\) concentration exceeded \(\geq\)0.25 mM, the cell transfection rates were reduced significantly even though cells were still viable. With 0.25 mM CaCl\({}_{2}\) and a viability of 28.8%, only 8.6% of cells were GFP-positive when the electric field of 1200 V/cm was applied.