# Efficient Gene Transfection by Electroporation--In Vitro and In Silico Study of Pulse Parameters
## 3 Results
### Time Dynamics of pDNA Expression  
The time dynamics of pDNA expression was also monitored for both cell lines for those pulse protocols with which we obtained the best overall GET with 500 \(\upmu\)g/mL of pEGFP-N1 plasmid (Table 1). The percentage of GFP-positive cells and their MFI were measured every 8 h for 6 days. Since we did not measure cell survival at each time point, the results for time dynamics of pDNA expression are presented as GET (not overall GET). Our results show that the dynamics of the onset of GFP expression (both percentage of GFP-positive cells and their MFI) are comparable for all pulse protocols tested (i.e., from 200 ns up to 5 ms pulse durations) but varied greatly between the two cell lines (Figures 10 and 11). Figure 9: Median fluorescence intensity (MFI) of GFP-positive cells with different pulse protocols for C2C12 myoblasts and 1306 fibroblasts with 500 \(\upmu\)g/mL of pEGFP-N1. **C2C12:** 25 \(\times\) 200 ns: 25 \(\times\) 200 ns, 10 Hz, 15.8 kV/cm; 100 \(\times\) 200 ns: 100 \(\times\) 200 ns, 10 Hz, 6.9 kV/cm; 300 \(\times\) 200 ns: 300 \(\times\) 200 ns, 10 Hz, 6.3 kV/cm; 25 \(\times\) 500 ns: 25 \(\times\) 500 ns, 10 Hz, 6.9 kV/cm; 100 \(\times\) 500 ns: 100 \(\times\) 500 ns, 10 Hz, 4.1 kV/cm; 300 \(\times\) 500 ns: 300 \(\times\) 500 ns, 10 Hz, 2.4 kV/cm; HF-BP: 2-2-2, 2-2, 32 pulses, 100 bursts, 1 Hz, 1.25 kV/cm; 8 \(\times\) 100 \(\upmu\)s: 8 \(\times\) 100 \(\upmu\)s, 1 Hz, 1.25 kV/cm; 8 \(\times\) 5 ms, 8 \(\times\) 5 ms, 1 Hz, 0.4 kV/cm. **1306:** 25 \(\times\) 200 ns: 25 \(\times\) 200 ns, 10 Hz, 12.8 kV/cm; 100 \(\times\) 200 ns: 100 \(\times\) 200 ns, 10 Hz, 6.9 kV/cm; 300 \(\times\) 200 ns: 300 \(\times\) 200 ns, 10 Hz, 4.2 kV/cm; 25 \(\times\) 500 ns: 25 \(\times\) 500 ns, 10 Hz, 6.5 kV/cm; 100 \(\times\) 500 ns: 100 \(\times\) 500 ns, 10 Hz, 3.5 kV/cm; 300 \(\times\) 500 ns: 300 \(\times\) 500 ns, 10 Hz, 2.7 kV/cm; HF-BP: 2-2-2, 32 pulses, 100 bursts, 1 Hz, 1 kV/cm; 8 \(\times\) 100 \(\upmu\)s: 8 \(\times\) 100 \(\upmu\)s, 1 Hz, 1.25 kV/cm; 8 \(\times\) 5 ms; 8 \(\times\) 5 ms, 1 Hz, 0.6 kV/cm. 0 V represents cells not exposed to electric pulses. Results are represented as an average of 3 repetitions. Bars represent standard deviation. Asterisk * represents statistically significant (\(p\) < 0.05) difference between cell lines for the same pulse protocol. In C2C12 myoblasts, the GET increased with all pulse protocols reaching a maximum at 32 h after GET. The best GET was achieved with the 8 \(\times\) 100 \(\upmu\)s pulse protocol (46.2%), followed by HF-BP (31.9%), 8 \(\times\) 5 ms (17.8%) and 25 \(\times\) 200 ns (6.9%). Afterwards the GET started to decline and after 144 h (i.e., 6 days) decreased below 10% for all pulse protocols reaching 4.5%, 0.7%, 1.1%, and 0.02% for the 8 \(\times\) 100 \(\upmu\)s, HF-BP, 8 \(\times\) 5 ms, and 25 \(\times\) 200 ns pulse protocols, respectively (Figure 10A). Figure 10: **(A)** GET and **(B)** median fluorescence intensity (MFI) of GFP-positive C2C12 myoblasts with 500 \(\upmu\)g/mL pEGFP-N1 pDNA with different pulse protocols on 1306 fibroblasts, 25 \(\times\) 200 ns: 25 \(\times\) 200 ns, 10 Hz, 12.8 kV/cm; HF-BP: 2-2-2-2, 32 pulses, 100 bursts, 1 Hz, 1.25 kV/cm; 8 \(\times\) 100 \(\upmu\)s: 8 \(\times\) 100 \(\upmu\)s, 1 Hz, 1.25 kV/cm; 8 \(\times\) 5 ms: 8 \(\times\) 5 ms, 1 Hz, 0.6 kV/cm, 0 V represents control where cells were not exposed to electric pulses. Results are represented as an average of 3 repetitions. Bars represent standard deviation. the HF-BP pulse protocol. The best GET was achieved with the 8 \(\times\) 100 \(\upmu\)s pulse protocol (max at 48 h, 55.2%), followed by the HF-BP (max at 80 h, 50.8%), 25 \(\times\) 200 ns (max at 40 h, 29.2%) and 8 \(\times\) 5 ms protocols (max at 40 h, 24.3%). Interestingly, higher GET was achieved with 25 \(\times\) 200 ns compared to 8 \(\times\) 5 ms protocol. After reaching a peak, the percentage of GFP-positive cells started to decrease slowly, except for the HF-BP protocol, where it remained at the same level for the entire time of observation.