# Nucleofection as an Efficient Method for Alpha TC1-6 Cell Line Transfection
## 3 Results and Discussion
### Nucleefection of \(\alpha\)TC1-6 Cells  
Furthermore, we used a nucleofection as an attractive alternative transfection method that could offer high efficiency of transfection level. Nucleofection is a type of electroporation that represents a widely used physical transfection method. As a result of an applied electrical pulse, transient pores are formed in the cell and nuclear membrane allowing for a free pass of target molecules [26]. In this way, nucleic acids enter directly into the nucleus, which increases the efficiency of transfection. This method provides faster gene expression because there is no need for cell division, which is very important for non-dividing primary cells. Figure 1: -Transfection of \(\alpha\)TC1-6 cells performed using PEI. Cells were transfected with mVenus plasmid vector in 1:3 = DNA:PEI ratio. (**A**) Cell viability and (**B**) percent of fluorescent cells was observed fifth day post-transfection (dpt) by flow cytometry. (**C**) Flow cytometric acquisition of \(\sim\)30% of GFP positive cells. (**D**) Representative images of efficient \(\alpha\)TC1-6 transfection on the 5 th dpt. Cell nuclei were stained with DAPI. The results are expressed as means \(\pm\) SDs. Significance between samples was determined by using an unpaired Students t-test, \({}^{*}p\leq 0.05\). Figure 2: PEI transfection of \(\alpha\)TC1-6 cells with the EpiCRISPR mix of plasmids. Cells were transfected with a plasmids mix: 5% Venus C1, 20% \(\alpha\)Cas9-Dmnt3a3L-KRAB, and 75% empty gRNA plasmid. (**A**) Fluorescent microscopy showed very few fluorescent cells on the fifth dpt. (**B**) Flow cytometry showed that there was \(\sim\)3.8% of transfected GFP positive cells. (**C**) Relative expression level of Cas9 mRNA in GFP positive sorted cells on the fifth dpt was measured by RT-qPCR. The results are expressed as means \(\pm\) SDs. Significance between samples was determined by using an unpaired Students \(t\)-test, \(***\)\(p<0.0001\). In our experiments, Nucleofector(r) Technology was the method of choice due to its higher efficiency for large DNA-fragment transfection and increasing cell survival rate in comparison with the electroporation [27; 28]. Also, nucleofection allows multiple samples to be transfected at the same time, making it a higher throughput method than electroporation. There was no ready-to-use optimized protocol or published data for \(\alpha\)TC1-6 cells using this transfection tool; therefore we start to analyze the best conditions for effective transfer of plasmid molecules in cells which involves a balance between the highest efficiencies of gene transfer and the minimal amount of cell death [19] (Figure 3). The specific combination of predefined pulses and three different solutions were used with the unknown supplement composition in combination with the pmaxGFP(tm) control vector, which encodes the green fluorescent protein from _Copepod Pontellina_ sp., all provided from Cell Line Optimization 4D-Nucleofector(tm) X Kit. The SF buffer stood out as the best among three different solutions (SE, SF, and SG) in combination with 15 different predefined Nucleofector(tm) programs. For this experiment, we used 0.4 ug of pmaxGFP(tm) for 2 \(\times\) 10\({}^{5}\)\(\alpha\)TC1-6 cells suspended in 20 \(\mu\)L SF, SG, or SE 4D-Nucleofector X Solution with a supplement for one well in 16-well Nucleovetute(tm) Strips as was recommended. After nucleofection, cells were immediately seeded in four 96-wells (0.5 \(\times\) 10\({}^{5}\) per well). Cells that were in SF buffer show the best percentage of viability, divided into two groups: one with high survival level and the other with a lower survival level. Compared to the other two buffers, the SF buffer had the least impact on cell membrane disruption, enabling most of the cells to survive and successfully recover after applied electrical pulses (Figure 3A). Also, these cells showed the best percentage of GFP positive cells (\(\sim\)50% GFP positive cells) (Figure 3B).