## 2 Materials and Methods
### Cell Harvesting and Electroporation  
For the electroporation experiments, the cells were harvested according to the standard procedure. Briefly, the growth medium was removed, the cells were washed with 1\(\times\) PBS (Sigma-Aldrich, St. Louis, MO, USA), and then the cells were incubated with Tryps-EDTA solution (Sigma-Aldrich, St. Louis, MO, USA) at 37 \({}^{\circ}\)C for 4 min. The cells were resuspended in an equal volume of medium, centrifuged for 2 min at 1000 rpm, and counted using a hemocytometer (Paul Marienfeld GmbH & Co. KG, Lauda-Konigshofen, Germany). Then, the required number of cells was mixed with a HEPES-based electroporation medium containing 10 mM HEPES (Lonza, Basel, Switzerland), 250 mM sucrose (Sigma-Aldrich, St. Louis, MO, USA), and 1 mM MgCl\({}_{2}\) (Sigma-Aldrich, St. Louis, MO, USA). The medium was prepared in Milli-Q water and then supplemented with different concentrations (0, 0.01, 0.1, 0.25, 0.5, or 1 mM) of CaCl\({}_{2}\) (Sigma-Aldrich, St. Louis, MO, USA). The concentration of cells in the sample was kept constant at 2 \(\times\) 10\({}^{6}\) cells/mL. All of the experiments were performed using 2 sets of electroporation parameters: 1400 V/cm for 100 us and 1200 V/cm for 250 us using a single square-wave electric pulse. The electrodes were composed of stainless steel with a 2 mm gap in between them. An electroporator (AmberCharge, Palanga, Lithuania) was used for the generation of electric pulses. The principal scheme of the study is provided in Figure 1.  
Electrotransfer of pDNA, Electrotransfer of Propidium Iodide (PI), and the Evaluation of Cell Viability  
For each experimental point, 45 uL of cell suspension (9 \(\times\) 10\({}^{4}\) cells) already supplemented with a different CaCl\({}_{2}\) concentration was mixed with 5 uL of the plasmid DNA solution to the final concentrations of 50, 100, 150, and 200 ug/mL pDNA in order to obtain a high electrotransfer efficiency with a minor effect on the cell viability [21]. After electroporation, the cells were placed in 24-well plates (TPP, Trasadingen, Switzerlands) for 15 min for recovery. After the recovery time, each well was supplemented with 950 uL of DMEM. Then, 24 h after electroporation, the growth medium was removed and cells were washed using 1\(\times\) PBS and resuspended using 1\(\times\) TrypIE Express (Thermo Fisher Scientific, Washington, DC, USA). After subsequent incubation for 10 min at 37 \({}^{\circ}\)C, the cells were subjected to analyses of flow cytometry and determination of gene electrotransfer (GET) efficiency, mean fluorescence intensity (MFI), and cell viability. GET was evaluated as the percentage of the viable GFP-positive cells in the population, and the MFI (mean fluorescence intensity) was analyzed in the fraction of GFP-positive cells. Both GET and MFI were then used in the calculation of the total fluorescence intensity:  
\[\text{Total fluorescence intensity}=\text{GET}*\text{MFI} \tag{1}\]  
For the evaluation of the cell viability, a flow cytometry assay (FCA) was used. The number of cells to collect was set to be constant at 1 \(\times\) 10\({}^{4}\) for all experimental points. Therefore, changes in the cell viability for each analyzed sample were reflected by changes in the  
Figure 1: The principal scheme of the experiments performed in this study in order to evaluate pDNA and PI electrotransfer dynamics at different concentrations of CaCl\({}_{2}\). volume required to collect this constant number of cells. The values for the control samples were normalized to 100%, and the cell viability in other samples was calculated accordingly.