EYFP Flow Cytometry Results

Flow Cytometry Report of Specimen 1
LNP Volume vs EYFP Delivery Efficiency
Data Summary
Sample # LNP (μL) % FITC-H Total Count
1 0 0.19 11295
2 5 0.69 11315
3 10 6.33 10732
4 20 14.76 8075
5 30 34.34 7195
6 50 32.20 8769
Analysis

The data above shows the results of our Flow Cytometry Data from transfections with EYFP plasmid in order to test the efficiency of our Hydrogel-LNP Matrix. Total concentration of Hydrogel at a constant 0.2% per the manufacturer instruction, and LNP volume was varied according to experimental plan. In total, we tested a set of six volumes ranging from 0 μL to 50 μL. After incubation, cells were then lifted and suspended in PBS before measuring the number of cells that showed a measure of fluorescence intensity. According to the data, increasing the volume of LNP solution used directly correlated to an increase in the transfection efficiency, with our highest candidate being 30 μL at 34.34% (which we used in all subsequent transfections). The efficiency then seemed to decrease or plateau, although we were unsure which. Interestingly, the total viable cell count taken by the machine varied (12000 total events per sample), decreasing until the 30 μL mark before increasing again. This suggests that the LNPs may induce some form of strain on the cell when delivering the gene, and thus, further experiments should be conducted to determine whether or not the LNPs have any measurable cytotoxic effect.

Western Blot Results

Western Blot results for dCas9 expression
Figure 1: The image above shows the four different plates transfected at various Dox timings. Each plate followed the same layout A—L only varying in Dox timing. A—D were all at a Dox concentration of 200 ng/mL, E—H at 500 ng/mL, and I—L at 1000 ng/mL. Columns A, E, and I possessed a 0.5:1 ratio of plasmid, B, F, and J had a 1:1 ratio, C, G, and K had a 1.5:1 ratio, and D, H, and L had a 0:1 ratio. The membrane was split into two halves for each protein (GAPDH at ~35 kDa and dCas9-VP64 at ~170 kDa). Resultant chemiluminescent blots are shown above.
Analysis

The WB above shows the four different plates transfected at various Dox timings. Each plate following the same layout as follows: A—D were all at a Dox concentration of 200 ng/mL, E—H at 500 ng/mL, and I—L at 1000 ng/mL. Columns A, E, and I possessed a ratio of 0.5 : 1 (for rtTA to dCas9), Columns B, F, and J had a 1 : 1 ratio, Columns C, G, and K had a 1.5 : 1 ratio, and lastly Columns D, H, and L had a 0 : 1 ratio (Cntrl). The membrane was split in half for each protein (GAPDH at ~35 kDa and dCas9-VP64 at ~ 170kDa). The above is after chemiluminescent HRP activation soln. was added to the blots, and imaged under UV light.

As shown in the data, the weak GAPDH signals completely invalidate the experimental soundness, necessitating a second round of work to be done. Although faint signals can be detected at a high mass, we determined that the signals present were around 200 kDa, much too large to be the dCas9-VP64 complex. This was further supported by the presence of protein in columns D/H/L, which lacked the rtTA plasmid (and should therefore be unable to induce dCas9 translation). Ultimately, the data is poor and inconclusive, and needs further revision (as of October 8th, 2025) to draw any sort of conclusions about the nature of the pTRE3G-dCas9-VP64-3xFLAG (NTD) plasmid.