# Efficient Gene Transfection by Electroporation--In Vitro and In Silico Study of Pulse Parameters
## 3 Results
### Effect of pDNA Concentration on GET, MFI, Cell Survival, and Overall GET  
In determining the effect of pDNA concentration on GET, MFI, cell survival, and overall GET, six different concentrations of pEGFP-N1 were tested, namely, 0, 40, 80, 100, 250, and 500 \(\upmu\)g/mL. The GET in C2C12 myotubes was significantly higher with HF-BP, 8 \(\times\) 100 \(\upmu\)s, and 8 \(\times\) 5 ms pulses compared to nanosecond pulses for all pDNA concentrations tested (Figure 3A). Interestingly, in 1306 fibroblasts, the GET was higher with HF-BP, 8 \(\times\) 100 \(\upmu\)s, and 8 \(\times\) 5 ms pulses compared to nanosecond pulses up to 250 \(\upmu\)g/mL of pDNA. However, at the highest pDNA concentration in 1306 fibroblast, there were no significant differences in the GET between all pulse protocols (Figure 3C). The effect of the pDNA concentration on GET depends on the cell line. However, the efficiency of nanosecond pulses depends more on the pDNA concentration compared to HF-BP, 8 \(\times\) 100 \(\upmu\)s, and 8 \(\times\) 5 ms pulses in both cell lines. The MFI of cells is indicative of how many pDNA copies were successfully transcribed and translated into fluorescent protein. The MFI of GFP-positive cells was higher in 1306 fibroblasts at all pDNA concentrations when compared to C2C12 myoblasts for all pulse protocols. The MFI of C2C12 myoblast was the highest with 8 \(\times\) 5 ms pulse protocol followed by 8 \(\times\) 100 \(\upmu\)s, HF-BP, and the lowest with nanosecond pulse protocols. Interestingly, the MFI of C2C12 myoblasts slowly increased up to 250 \(\upmu\)g/mL of pDNA and then reached a plateau. For pulse protocols 8 \(\times\) 5 ms and 8 \(\times\) 100 \(\upmu\)s, there was a small increase in MFI from 250 \(\upmu\)g/mL to 500 \(\upmu\)g/mL pDNA concentration, but not statistically significant (Figure 3B). Similarly, MFI of 1306 fibroblasts was also different compared to C2C12 myoblasts. At the highest pDNA concentration, the MFI of 1306 fibroblasts was the highest with the 8 \(\times\) 100 \(\upmu\)s pulse protocol followed by the nanosecond, HF-BP, and 8 \(\times\) 5 ms pulse protocols. The MFI after the HF-BP and 8 \(\times\) 5 ms pulse protocols increased gradually with increasing pDNA concentration. With the 8 \(\times\) 100 \(\upmu\)s pulse protocol, a significant increase was observed between 100 \(\upmu\)g/mL and 250 \(\upmu\)g/mL pDNA concentration. Interestingly, the MFI of 1306 fibroblasts increased significantly from 250 \(\upmu\)g/mL to of 500 \(\upmu\)g/mL pDNA concentration with both 200 ns and 500 ns pulse protocols (Figure 3D). The MFI of GFP-positive cells is cell-line-dependent and increases with increasing pDNA concentration. \begin{table}
\begin{tabular}{c c} \hline
**C2C12 (16.8 \(\pm\) 1.8 \(\upmu\)m)** & **1306 (14.3 \(\pm\) 1.4 \(\upmu\)m)** \\ \hline
25 \(\times\) 200 ns, 10 Hz, 15.8 kV/cm & 25 \(\times\) 200 ns, 10 Hz, 12.8 kV/cm \\ \hline
100 \(\times\) 500 ns, 10 Hz, 4.1 kV/cm & 25 \(\times\) 500 ns, 10 Hz, 6.5 kV/cm \\ \hline
2-2-2-2, 32 p, 100 b, 1 Hz, 1.25 kV/cm & 2-2-2-2, 32 p, 100 b, 1 Hz, 1 kV/cm \\ \hline
8 \(\times\) 100 \(\upmu\)s, 1 Hz, 1.25 kV/cm & 8 \(\times\) 100 \(\upmu\)s, 1 Hz, 1.25 kV/cm \\ \hline
8 \(\times\) 5 ms, 1 Hz, 0.4 kV/cm & 8 \(\times\) 5 ms, 1 Hz, 0.6 kV/cm \\ \hline \end{tabular}
\end{table} 
Table 1: Pulse protocols used to determine the effect of pDNA concentration. High concentration of pDNA used for GET was reported to affect cell survival [16]. In our study, however, with 8 \(\times\) 5 ms pulses, we did not observe any decrease in cell survival with increasing pDNA concentrations compared to cells that were exposed just to electric pulses in both cell lines. In addition, in C2C12 myoblasts, pDNA concentration did not have any effect on cell survival after GET with 500 ns and 200 ns pulse protocols. With the 8 \(\times\) 100 \(\upmu\)s pulse protocol in C2C12, survival was significantly lower, with 500 \(\upmu\)g/mL of pDNA.