## 3 Results
A similar tendency appeared with the electric field of 1400 V/cm: only 3.1% of cells were transfected, and viability was still above 20%. A further increase in the CaCl\({}_{2}\) concentration to 0.5-1 mM further confirmed these observations and revealed that even though -20% of cells were still viable, no transfection was detected for either electroporation protocol. These results suggested that the reduced GET efficiency was not solely the result of cell death, and that secondary mechanisms were implicated to some extent. Figure 3: The effect of different CaCl\({}_{2}\) concentrations on cell viability (**A,B**), overall GET (**C**), and pDNA electrophoretic mobility (**D**). For panels A, B, and C, cells were treated using one square pulse of 1200 V/cm for 250 μs or 1400 V/cm for 100 μs. 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 differences between calcium electroporation alone (pink squares) and in combination with pDNA (green triangles) at different CaCl\({}_{2}\) concentrations are denoted by *, **, and *** (Panels A and B). For panel C, 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 *** (1200 V/cm) and *** (1400 V/cm). * represents the \(p\) < 0.005, **—\(p\) < 0.01 and *** or *** represents \(p\) < 0.001. For panel D, pDNA was electroporated using a single 1200 V/cm, 250 μs electric pulse; and electrophoretic mobility was evaluated via agarose gel electrophoresis. Since Ca\({}^{2+}\) can bind to DNA [23], we decided to investigate whether the observed phenomenon was the result of the altered electrophoretic mobility of pDNA; e.g., due to pDNA aggregation. For this experiment, the plasmid was mixed with electroporation medium containing different concentrations of CaCl\({}_{2}\), and agarose gel electrophoresis subsequently was performed. As presented in Figure 3D, there was no significant relationship observed between the pDNA mobility and the Ca\({}^{2+}\) concentration in the sample. As shown previously, pDNA electrotransfection induced a significant cytotoxic effect on the cell viability when CaCl\({}_{2}\) was present before electroporation. In order to better understand the Ca\({}^{2+}\)-induced decrease in transfection efficiency, cell death induced by calcium electroporation alone and by pDNA electrotransfer alone (Figure 4A,B) were evaluated separately. In addition to previous observations, the data revealed that the number of cells that had died due to the impact of pDNA increased with an increasing CaCl\({}_{2}\) concentration. From 5.9% at 0 mM CaCl\({}_{2}\) in the medium, the proportion of pDNA-related cell deaths increased to 37.6% at 0.25 mM CaCl\({}_{2}\) when an electric field of 1200 V/cm was used (\(p<0.001\)). A similar tendency was observed when an electric field of 1400 V/cm was applied: the pDNA-related cell death increased from 17.6% (0 mM) to 56.9% (0.25 mM) (\(p<0.001\)) Since electroporation alone--with 0.5-1 mM CaCl\({}_{2}\) pre-administered to the medium--was responsible for a significant decrease in the cell viability, the effect observed previously was no longer detected. In order to gain a deeper understanding of the intracellular processes related to Ca\({}^{2+}\) electroporation, a PI assay was performed. PI was added at two different time points: before or 15 min after pulse application with 0-1 mM CaCl\({}_{2}\) present in the medium. As illustrated in Figure 5A, the increase in calcium concentration was related to the decrease in the percentage of PI-positive cells. This phenomenon indicated that calcium could enhance the rate of pore resealing, which we previously demonstrated [24; 25].