# 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
Interestingly, with HF-BP pulses in C2C12 myoblasts, a significant decrease in cell survival was observed already with the lowest pDNA concentration (40 \(\upmu\)g/mL). In 1306 fibroblasts with the 500 ns and 200 ns pulse protocols, survival was significantly lower, with 500 \(\upmu\)g/mL of pDNA. With HF-BP pulses with concentrations higher than 100 \(\upmu\)g/mL and with the 8 \(\times\) 100 \(\upmu\)s pulse protocol, survival was significantly lower with 250 \(\upmu\)g/mL and 500 \(\upmu\)g/mL of pDNA (Figure 4). Our results suggest that the decrease in survival after GET with high pDNA concentrations is cell-line-dependent. Figure 3: GET and median fluorescence intensity (MFI) of GFP-positive cells with different concentrations of pEGFP-N1 pDNA. (**A**) GET of C2C12 myoblasts, pulses: 25 \(\times\) 200 ns: 25 \(\times\) 200 ns, 10 Hz, 15.8 kV/cm; 100 \(\times\) 500 ns: 100 \(\times\) 500 ns, 10 Hz, 4.1 kV/cm; HF-BP: 2-2-2-2, 32p, 100b, 1 Hz, 1.25 kV/cm; 8 \(\times\) 100 \(\upmu\)s: 8 \(\times\) 100 \(\upmu\)s, 1 Hz, 1.25 kV/cm; and 8 \(\times\) 5 ms: 8 \(\times\) 5 ms, 1 Hz, 0.4 kV/cm. (**B**) MFI of C2C12 myoblasts, pulses: 25 \(\times\) 200 ns: 25 \(\times\) 200 ns, 10 Hz, 15.8 kV/cm; 100 \(\times\) 500 ns: 100 \(\times\) 500 ns, 10 Hz, 4.1 kV/cm; HF-BP: 2-2-2, 32p, 100b, 1 Hz, 1.25 kV/cm; 8 \(\times\) 100 \(\upmu\)s: 8 \(\times\) 100 \(\upmu\)s, 1 Hz, 1.25 kV/cm; and 8 \(\times\) 5 ms: 8 \(\times\) 5 ms, 1 Hz, 0.4 kV/cm. (**C**) GET of 1306 fibroblasts, pulses: 25 \(\times\) 200 ns: 25 \(\times\) 200 ns, 10 Hz, 12.8 kV/cm; 25 \(\times\) 500 ns: 25 \(\times\) 500 ns, 10 Hz, 6.5 kV/cm; HF-BP: 2-2-2, 32p, 100b, 1 Hz, 1.8 kV/cm; and 8 \(\times\) 5 ms: 8 \(\times\) 5 ms, 1 Hz, 0.6 kV/cm. (**D**) MFI of 1306 fibroblasts, pulses: 25 \(\times\) 200 ns: 25 \(\times\) 200 ns, 10 Hz, 12.8 kV/cm; 25 \(\times\) 500 ns: 25 \(\times\) 500 ns, 10 Hz, 6.5 kV/cm; HF-BP: 2-2-2, 32p, 100b, 1 Hz, 1.8 kV/cm; 8 \(\times\) 100 \(\upmu\)s: 8  
control where cells were not exposed to electric pulses. Results are represented as an average of 3 repetitions. Statistical differences are given in the text. Bars represent standard deviation. When also considering cell survival, i.e., calculating overall GET, we obtained the results presented in Figure 5. The overall GET increased with the increasing pDNA concentration for all pulse protocols and both cell lines. In C2C12 myoblasts, the highest overall GET was reached with 8 \(\times\) 5 ms and 8 \(\times\) 100 us pulse protocols, followed by HF-BP, 500 ns, and 200 ns protocols. With all five pulse protocols, the overall GET increased up to the highest pDNA concentration used, i.e., 500 ug/mL. In C2C12 myoblasts, the overall GET with 40, 80, and 100 ug/mL pDNA concentrations was, however, below 1% with both 200 ns and 500 ns pulses (Figure 5A). In 1306 fibroblasts, the highest overall GET was observed with 8 \(\times\) 100 us pulse protocol regardless of pDNA concentration used. Interestingly, with the highest pDNA concentration, i.e., 500 ug/mL, using the 8 \(\times\) 5 ms pulse protocol, we achieved the lowest overall GET, even lower than the nanosecond and HF-BP pulse protocols. In addition, the overall GET increase with the increasing pDNA concentration was different compared to C2C12 myoblasts. In 1306 fibroblasts with the 8 \(\times\) 100 us, 8 \(\times\) 5 ms, and HF-BP  
Figure 4: Cell survival with different concentrations of pEGFP-N1 pDNA.