## 2 Materials and Methods  
Cell culture: Chinese Hamster Ovary (CHO-K1) cells were grown as a monolayer at 37 \({}^{\circ}\)C in a 5% CO\({}_{2}\) humidified incubator and maintained in Dulbecco's modified Eagle's medium supplemented with 1% L-glutamine, 10% fetal bovine serum (Sigma, St Louis, MO, USA) and 1% penicillin-streptomycin solution (100 U/mL penicillin and 100 ug/mL streptomycin). Trypan Blue Stain exclusion test (Invitrogen, Carlsbad, CA, USA) was used to measure the cell viability during passaging. Cells were passaged 24 h before the experiments and every 2-3 days for routine passaging. Plasmid DNA: The pMAX-GFP plasmid DNA (Amaxa, Cologne, Germany) (3485 bp) was propagated in E. coli DH5\(\alpha\) and purified using the EndoFree Plasmid GigaPrep kit (Qiagen, Chatsworth, CA, USA) according to the manufacturer's instructions. The extracted plasmid DNA was validated by electrophoresis on agarose gel. The concentration was determined by measuring the absorbance at 260 nm in a spectrophotometer (Nanodrop 2000, Thermo Fisher, Washington, DC, USA). Electroporation medium and electroporator: Electroporation medium was composed of sucrose (242 mM), Na\({}_{2}\)HPO\({}_{4}\) (5.5 mM), NaH\({}_{2}\)PO\({}_{4}\) (3 mM) and MgCl\({}_{2}\) (1.7 mM). Electroporation medium was isosmotic (270 mOsm) and had 7.1 pH. The measured specific conductivity of electroporation medium was 0.1 S/m at 25 \({}^{\circ}\)C.  
During electroporation, 1, 2, or 3 square-wave high voltage (HV) pulses (1400 V/cm pulse strength, 100 \(\upmu\)s pulse duration, 1 Hz repetition frequency) were delivered using electroporator (BTX T820, Harvard Apparatus, Holliston, MA, USA). Experiments were performed with stainless steel plate electrodes produced in Biophysical research laboratory, Vytautas Magnus University. The fixed distance of 2 mm between the electrodes was used for all experiments. DNA electrotransfer and result measurement procedure: During the experiment, cells were trypsinized, centrifuged (2 min at 170 RCF) and suspended in low conductivity electroporation medium at a concentration of 2 million cells per milliliter. For each experimental point, 45 \(\upmu\)L of cell suspension containing 9 \(\times\) 10\({}^{4}\) cells were used, and 5 \(\upmu\)L of plasmid solution was added to the sample to get final plasmid concentration of 10-600 \(\upmu\)g/mL. The resulting solution (50 \(\upmu\)L) was electroporated and removed by the gentle tap of the electrodes at the bottom of 24 well plate. Then the cells were incubated for 10 min to reseal the electroporated cell membrane. Afterwards, the treated cells were supplemented with 950 \(\upmu\)L of growth medium. Then, 900 \(\upmu\)L of cell suspension was put into the incubator (37 \({}^{\circ}\)C, 5% CO\({}_{2}\)) for 24 h in 24 well tissue culture plate (TPP, Trasadingen, Switzerland). After 24 h, cells we are trypsinized, centrifuged and resuspended in 100 \(\upmu\)L PBS for flow cytometry (BD Accuri C6, BD Biosciences, Franklin Lakes, NJ, USA) measurements. 1 \(\times\) 10\({}^{4}\) cells were gathered from each experiment. For all measurements, flow rate was kept at 66 \(\upmu\)L/min and the core size was 22 \(\upmu\)m. The cells were excited using 488 nm (blue) laser, and the fluorescence was collected using 533/30 nm bandpass filter. The results were analyzed with BD Accuri C6 Software, obtaining the percentage of transfected cells (referred to as transfection efficiency (_TE_)) and mean cell fluorescence (_MCF_) of transfected cell population. Total fluorescence (_TF_) was calculated using Equation (1):  
\[TF=\frac{TE*10000*MCF}{100} \tag{1}\]  
Cell viability was measured using the clonogenic assay. After the 10 min incubation and addition of 950 \(\upmu\)L of growth medium, 100 \(\upmu\)L of cell suspension was taken and put in 900 \(\upmu\)L of growth medium, and 44.4 \(\upmu\)L of this suspension (\(\sim\)400 cells) was placed in 40 mm diameter Petri dish containing 2 mL of growth medium.