# Efficient Gene Transfection by Electroporation--In Vitro and In Silico Study of Pulse Parameters
## 3 Results
### Determining Optimal Electric Field Amplitude for GET  
In order to determine the optimal parameters for GET (intersections of permeabilization and survival curves) for C2C12 myoblast and 1306 fibroblast cell lines, we varied the nanosecond pulse parameters--pulse duration: 200 or 500 ns; pulse number: 25, 100, or 300 pulses; pulse repetition frequency: 5 or 10 Hz, and delivered electric field: 0-19 kV/cm. In both cell lines, C2C12 myoblasts and 1306 fibroblasts, we achieved >90% cell membrane permeabilization, with different pulse durations of 200 ns and 500 ns and different tested pulse numbers, 25, 100, and 300 pulses (Supplementary Figures S2 and S3). No significant difference in permeabilization, survival, and PI MFI curves was observed when changing pulse repetition frequency (5 or 10 Hz) for 200 ns and 500 ns pulses for all three pulse numbers tested in C2C12 myoblasts (data not shown). Based on this, we decided to use only a 10 Hz pulse repetition frequency for experiments with 1306 fibroblasts. Intersections of permeabilization and survival curves were observed at similar electric fields for C2C12 myoblasts and 1306 fibroblasts, except for 25 \(\times\) 200 ns pulses, where the intersection of permeabilization and survival curves was observed at a lower electric field in 1306 cells--12-14 kV/cm compared to 15-18 kV/cm for C2C12 myoblasts (Supplementary Figure S2). Since cell size can influence the permeabilization threshold, we measured the diameters of cells in suspension, which were 16.8 \(\pm\) 1.8 \(\upmu\)m for C2C12 myoblasts and 14.3 \(\pm\) 1.4 \(\upmu\)m for 1306 fibroblasts. A higher MFI of PI-positive cells was observed with 500 ns pulses compared to 200 ns duration pulses; additionally, the MFI increased with the increasing number of pulses from 25 to 300 in both pulse durations tested. Comparing MFI between C2C12 myoblasts and 1306 fibroblasts, lower MFI was observed in 1306 fibroblasts with both 200 ns and 500 ns pulses but only at high electric field amplitudes (Supplementary Figure S4). The HF-BP pulse protocol consisted of bipolar pulses of 2 \(\upmu\)s duration of positive and negative phase, 2 \(\upmu\)s pause between positive and negative pulse phase, and pause between bipolar pulses. We applied 100 bursts, each containing 32 pulses. The burst repetition rate was 1 Hz, and the applied electric field was 0-2.5 kV/cm. For microsecond pulses we delivered: 8 \(\times\) 100 \(\upmu\)s, 1 Hz pulse repetition frequency, with the applied electric filed, 0-2 kV/cm and for millisecond pulses; 8 \(\times\) 5 ms, 1 Hz pulse repetition frequency and applied electric filed, 0-1.25 kV/cm. Permeabilization and survival curves for HF-BP pulse protocol behave similarly as permeabilization and survival curves of 8 \(\times\) 100 \(\upmu\)s pulse protocol for both cell lines. Permeabilization was achieved at lower values of electric field for 8 \(\times\) 5 ms compared to 8 \(\times\) 100 \(\upmu\)s pulse protocol and survival deceased at lower values of electric field for 8 \(\times\) 5 ms compared to 8 \(\times\) 100 \(\upmu\)s pulse protocol in both cell lines. MFI of permeabilized cells was higher at lower values of electric field when longer millisecond pulses were applied. Similarly, as for nanosecond pulses permeabilization, the MFI of permeabilized cells with a high electric field with HF-BP, micro-, and millisecond pulses was lower in 1306 cells compared to C2C12 myoblasts (Supplementary Figures S5 and S6). After the initial parameters were determined, based on the permeabilization and survival curve, further optimization for every pulse protocol was performed. PerformingGET, we tested three different electric field amplitudes in the range of slightly below to slightly above the intersection of permeabilization and survival curve to determine the electric field at which the overall GET was the best (data not shown).