## 4 Discussion  
Ca\({}^{2+}\) is one of the most prevalent and important compounds in both intracellular and extracellular environments. It is estimated that the intracellular concentration of Ca\({}^{2+}\) is in the micromolar range (0.1-0.2 \(\upmu\)M), whereas extracellular calcium concentration varies between 1 and 3 mM [26]. Therefore, it is not surprising that Ca\({}^{2+}\) influences various processes such as gene electrotransfer [20; 24; 25; 27; 28; 29]. The inhibitory effect of Ca\({}^{2+}\) on electrotransfer efficiency and luciferase fluorescence intensity has been observed in studies conducted in vivo while using relatively high CaCl\({}_{2}\) concentrations that varied between 10 mM and 1 M [20] However, when considering the pronounced difference in intra- and extracellular concentration, it is possible that even small alterations in the amount of Ca\({}^{2+}\) may induce significant impacts on diverse cellular processes. In the current study, we investigated the impact of low (0.01-1 mM) concentrations of CaCl\({}_{2}\) on the efficiency of pDNA electrotransfer using several electroporation protocols. The  
Figure 6: The dependence of pDNA electrotransfer efficiency (**A**), GFP total fluorescence intensity (**B**), cell viability (**C**), and normalized electrotransfer efficiency (**D**) on pDNA and CaCl\({}_{2}\) concentrations in the electroporation medium. Cells were treated using one square pulse of 1200 V/cm for 250 \(\upmu\)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 differences between the control (0 mM) and the 0.25, 0.5, and 1 mM CaCl\({}_{2}\) at different pDNA concentrations used are denoted by ***, ***, and \(\cdots\), respectively; three symbols denote \(p<0.001\). obtained results clearly demonstrated a decreased transfection efficiency with an increasing CaCl\({}_{2}\) concentration. The results revealed that a low CaCl\({}_{2}\) concentration of 0.25 mM pre-administered to the electroporation medium reduced the electrotransfer efficiency of the pDNA molecules by \(\sim\)1.5-fold (Figure 2A,B) and altered the cell viability in a concentration-dependent manner (Figure 3A,B). The data also suggested that the decrease in GET was not solely the result of a decrease in the number of viable cells. The results demonstrated that a higher CaCl\({}_{2}\) concentration was related to a lower fraction of GFP-positive cells being present in the viable population. When the CaCl\({}_{2}\) concentration was \(>\)0.25 mM, the percentage of GFP-positive cells in the viable population remained similar (\(\sim\)20%), but a gradual decrease in GET was still observed; this suggested the influence of secondary Ca\({}^{2+}\)-related mechanisms that affected GET. In this context, we speculated that gradual Ca\({}^{2+}\)-related energy depletion can affect many endocytotic, pDNA intracellular migration, transcription, and translation pathways needed for cell electrotransfection. The observed phenomenon led to two hypothesized mechanisms. One is that calcium modifies pDNA properties that are essential for efficient electrotransfer, thereby resulting in a reduced efficiency of the electrotransfer of pDNA. This hypothesis arose from studies that showed the neutralization of DNA charge induced by Ca\({}^{2+}\) and therefore reduced electrophoretic forces during electroporation [18; 23] One of these studies also proposed that pDNA has binding sites for Ca\({}^{2+}\) and is even able to compete with calmodulin for the binding of calcium [23]. In order to test the latter assumption, we performed agarose gel electrophoresis (Figure 3D); however, no difference in pDNA electrophoretic mobility was observed within the range of CaCl\({}_{2}\) concentration (0-1 mM) tested.