# Nucleofection as an Efficient Method for Alpha TC1-6 Cell Line Transfection
## 3 Results and Discussion
### Nucleefection of \(\alpha\)TC1-6 Cells
Transient pores generated after nucleofection remain open for about 15 min, enabling calcium ions from the DMEM medium containing high levels of calcium ions to enter the cell. This calcium influx could affect cell viability through the initiation of  
Figure 4: Comparison of the most potent combinations of SF 4D-Nucleofector X Solutions and nucleofection programs for \(\alpha\)TC1-6 cells. (**A**) The three most potent programs at 24 h after transfection are selected for monitoring on the sixth and eighth days after nucleofection. Control represents non-transfected cells. (**B**) Post-nucleofection time course of GFP expression level is monitored by using fluorescent microscopy. Representative photos of selected programs for nucleofection are shown. (**C**) The results of quantification of the fluorescent signal are expressed as means \(\pm\) SDs. Significance between groups was determined by using one-way ANOVA followed by [PERSON]’s post hoc test. *** \(p\) < 0.0001 compared to CM-137, *** \(p\) < 0.0001 compared to CM-150. many signal transduction pathways. RPMI medium with a low level of calcium ions was used as a \"recovery medium,\" supporting the cell's survival. After increasing the cell number for nucleofection by 75%, cell viability was measured 24 h after transfection. The viability of cells exposed to the program CM-137 were 17% lower if compared with the previous experiment (Figure 3A). The viability of the cells subjected to program CM-150 stood nearly the same as previously (89.9%) and showed a statistically significant higher rate of survival compared to the CM-137 program (Figure 5B). The additional recovery step after the electrical pulse had a beneficial effect on the increasing pull of transfected cells by increasing the number of GFP-positive cells by 8.6% for cells exposed to program CM-150, whereas there were no significant changes for cells exposed to program CM-137 (Figure 5C) compared to the previous experiment (Figure 3B). Our results revealed that nucleofection is a method of choice for \(\alpha\)TC1-6 cells transfection, because we obtained a better transfection rate and higher cell viability for \(\alpha\)TC1-6 cells in compare to the DNA:PEI transfection method (Figures 1 and 2). The next step in nucleofection experiments included the use of the EpiCRISPR mix of plasmids: 5% pmaxGFP(tm) (as a reporter gene), 20% dCas9-Dnmt3 aL-KRAB plasmid (as effector domain), and 75% empty gRNA plasmid in two different final amounts (0.5 \(\upmu\)g and 1 \(\upmu\)g) for 3.5 \(\times\) 10\({}^{5}\) cells per pulse (Figure 6). Twenty-four hours after the nucleofection, flow cytometry analysis showed that doubling the amount of EpiCRISPR mix of plasmid DNA did not affect the viability of the cells exposed to both programs CM-137 and CM-150 (Figure 6A). The cell survival rate was the same as in the previous experiment (Figure 4A) confirming the reproducibility of this tool for cell transfection. Despite the high degree of cell viability, transfection efficiency was affected by the size of plasmids (Figure 6B) as was noticed with DNA:PEI transfection (Figures 1 and 2). The pmaxGFP(tm) vector belongs to the group of the small reporter plasmids and is 3486 bp in size, whereas dCas9-Dnmt3a3L-KRAB is almost 3\(\times\) larger (11,548 bp in size). During the transfection process, larger plasmids can become tangled into open membrane pores, aggravating proper plasmid uptake and membrane reclosure and contributing to cell death [29]. As shown in Figure 6B, transfection efficiency was satisfying but lower than in previous experiments for 27.3% if CM-137 was applied and 33.6% if CM-150 was applied, because of co-transfection of \(\alpha\)TC1-6 cells with a mix of larger plasmids (5% pmaxGFP(tm), 20% dCas9-Dnmt3a3L-KRAB plasmid and 75% empty gRNA plasmid). As stated by others and similar to what we observed, through co-traveling across the cell and nuclear membrane, small plasmids facilitate the entrance of larger plasmids like  
Figure 5: Increased cell number enhances nucleofection outcomes.