# Nucleofection as an Efficient Method for Alpha TC1-6 Cell Line Transfection
## 3 Results and Discussion
### Nucleefection of \(\alpha\)TC1-6 Cells
(**A**) Cell viability and fluorescence were observed 24 h after seeding by flow cytometry. (**B**) Transfection efficiency was estimated as percentage of fluorescent cells. Nucleofection was performed with two different plasmid concentrations. The results are expressed as means \(\pm\) SDs. Significance between samples was determined by using an unpaired Students \(t\)-test. \({}^{**}p\leq 0.01\), \({}^{***}p<0.0001\). used \(5\times 10^{6}\) cells per NuclecuvetteTM. The nucleofection shows much better results in transfection efficiency than chemical DNA:PEI transfection for the analyzed cell line. Fluorescent microscope analysis showed the variable expression levels of the GFP at several time points indicating time-limited transient transfection of \(\alpha\)TC1-6. Ten days after nucleofection GFP is still detectable in the cells, but at a very low level compared to the other dpts (Figure 8A). Cells were analyzed and sorted by FACS on the fifth and seventh  
Figure 8: Monitoring of GFP expression level in cells nucleofected with selected CM-156 program. (**A**) Post-nucleofection time course of GFP expression level was monitored via fluorescent microscopy. Cell nuclei are stained with DAPI. (**B**) Flow cytometry analysis showed a high level of GFP expression on the fifth day after nucleofection, followed by a reduction of GFP expression on the tenth post-nucleofection day. The results are expressed as means \(\pm\) SDs. The significance between samples was determined by using an unpaired Student \(t\)-test,* \(p\leq 0.05\). Figure 7: Fine-tuning of the selected pulse. Transfection efficiency was observed 48 h after seeding \(5\times 10^{5}\) by fluorescent microscopy (**A**) and by flow cytometry on the fifth post-transfection day (**B**). (**C**) In an attempt to increase the number of GFP positive cells, the CM-150 and CM-156 program have been moved in a direction toward increasing efficiency. Fine-tuning matrix available at [[https://bioscience.lonza.com/download/content/asset/31152](https://bioscience.lonza.com/download/content/asset/31152)]([https://bioscience.lonza.com/download/content/asset/31152](https://bioscience.lonza.com/download/content/asset/31152)) (accessed on 4 February 2022). The results are expressed as means \(\pm\) SDs. The significance between samples was determined by using an unpaired Students \(t\)-test,* \(p\leq 0.05\). days after transfection. The detected level of GFP-positive cells was around 71.12% for the fifth and 59.5% for the seventh dpt (Figure 8B). The drop in the percentage of positive cells between the fifth and seventh day is a consequence of losing the plasmids during ongoing cell division and their degradation. The main function of alpha pancreatic cells is a secretion of glucagon which participates in maintaining glucose homeostasis [32]. After transfection, there were no differences in the presence of glucagon in the group of transfected cells relative to the control (untransfected) \(\alpha\)TC1-6 cells (Figure 9A). This indicates that the presence of the EpiCRISPR system in the transfected cells without gRNA for targeting the region of interest did not affect their main function. The relative level of Cas9 mRNA confirmed successful entrance and presence of EpiCRISPR system in transfected and collected GFP-positive \(\alpha\)TC1-6 cells at the fifth and seventh dpt (Figure 9B). The change in expression of the Cas9 mRNA level between two analyzed days is correlated with the change in expression of green fluorescent protein in transfected cells, demonstrating transient transfection of \(\alpha\)TC1-6 cells. The same was observed by [PERSON] and [PERSON], who noticed the specific growth rate was in correlation with the rates of the decrease of fluorescence in transiently transfected CHO cells [33]. Different transfection methods and reagents have to be defined for cells that are hard to transfect. The CRISPR/Cas system could be delivered to target cells by using numerous methods for cell transfection [34]. [PERSON] et al.