# Nucleofection as an Efficient Method for Alpha TC1-6 Cell Line Transfection
## 3 Results and Discussion
### Nucleefection of \(\alpha\)TC1-6 Cells
On the other hand, the SE buffer showed the worst results concerning \(\alpha\)TC1-6 cells' viability, resulting in a low number of transfected cells. Based on this result, in the next optimization experiment we used exclusively SF 4D-Nucleofector X Solutions (Figure 4). By using available recommendations, we increased the number of cells per transfection and included a recovery step with incubation in a low calcium medium (RPMI) for 10 min at 37 \({}^{\circ}\)C after nucleoporation. Figure 3: Comparison of three 4D-Nucleofector X Solutions and predefined programs for \(\alpha\)TC1-6 cells nucleofection. (**A**) Percentage of cell viability 24 h after nucleofection was observed by flow cytometry. Cells with no program were taken as a non-transfected control cells and were 100% viable. Percentage of nucleofected cells is expressed relative to control cells. (**B**) Percentage of GFP positive cells 24 h after nucleofection was observed by flow cytometry. Programs CM-137 and CM-150 were chosen between four programs (CA-137, CM-138, CM-137, CM-150) with the ability to provide the highest percentage of cell viability (around 80%), where CM-137 provided the highest and CM-150 the lowest transfection efficiency. Among the other 11 programs that induced lower viability after transfection (around 58%), DN-100 was chosen as a program providing the highest efficiency and very good survival rate (Figure 3). Cells exposed to CM-137 and CM-150 programs showed a similar rate of survival 24 h after nucleoporation whereas program DN-100 caused larger cell damage affecting the cell viability by 41.51% (Figure 4A). On the other hand, CM-137 and DN-100 programs gave a similar rate of transfection efficiency (CM-137-57%, DN-100-59%) whereas the CM-150 program induced a slightly lower efficiency rate (44.5%). GFP expression levels were examined on the sixth and eighth days after transfection for three selected pulses. The maintenance of GFP expression was noticed until cells were tracked (a few GFP positive cells on 14 dpt, data not shown). The cells exposed to the CM-150 program showed the best viability 24 h after nucleoporation and had the most successful recovery after stress caused by an electrical pulse (Figure 4B,C). This was indicated with initial low mortality after electrical pulse and level of cell divisions after nucleofection, followed by maintaining a high level of GFP fluorescence. Both CM-137 and CM-150 programs resulted in significantly more cells expressing GFP than the DN-100 program (Figure 4C), which highlights the importance of low cytotoxicity of the transfection method for obtaining sufficient pull of transfected cells for further analysis. Further optimization experiments were directed toward the increasing pull of transfected cells. There were two directions: one involved increase in the initial cell number for transfection and the other an increase of the amount of plasmid DNA (Figure 5). In order to increase the number of transfected cells, the effect of cell seeding density on cell viability was examined (Figure 5). As shown in Figure 3, 2 \(\times\) 10\({}^{5}\) cells were taken as 100% viable. We observed that doubling the cells' number had no adverse effect on cell viability 24 h after seeding. An additional significant increase of cell numbers harmed cell viability because high initial cell densities might have a negative effect on cell metabolism and nutrient availability (Figure 5A). Furthermore, nucleofection optimization included a comparison of CM-137 and CM-150 programs in order to choose the most appropriate transfection conditions (Figure 5B,C). For each program, we used 3.5 \(\times\) 10\({}^{5}\)\(\alpha\)TC1-6 cells and 0.5 \(\upmu\)pmaxGFP\({}^{\text{TM}}\) plasmid. Cells were suspended in 20 \(\upmu\)L SF 4D-Nucleofector X Solutions, including incubation in RPMI medium for 10 min after nucleoporation at 37 \({}^{\circ}\)C as a recovery step. This recovery step was included to prevent calcium influx from media to the cell.