4. Summary of Experiments
4.3 Poly-D-Lysine Qualification 2
The primary goal of this experiment was to test different concentrations and incubation times to see which conditions would result in a higher percent of dead cells. It was found that there were roughly the same number of live cells in the pilot study when comparing between a coated and uncoated PDL culture plate. To find out if the coating held on to more dead cells, the percent dead was used as the defining parameter. Again, total dead cell count could not be used
coverage between images. The dead cell count was normalized to the total cells present in each image, as a percent.
The secondary goal was introducing ultraviolet radiation to make sure UV did not affect the coating. Testing this parameter will be merely qualitative and acknowledging adverse effects.
The experimental setup was comprised of four 8-well cell culture plates. Plate 1 was a control with no coating. Plate 2 had a concentration of 100ug/ml and was incubated for 5 minutes, the same as the pilot study. Plate 3 had the same concentration of 100ug/ml, but was incubated for 30 minutes. The final plate had a concentration of 200ug/ml, the stock solution, with a 30-minute incubation time. All plates were dried for at least 2 hours before seeding keratinocytes. One day after a 35mJ exposure of UV, the cells were fixed with PFA and stained with Ethidium homodimer-1 to detect dead cells, and Hoechst to detect all cells.
Image acquisition consisted of taking three 10x stitched images of varying depths in the middle of each well. Each stitched image was a 3x3 matrix of 10X images, while depth varied 4um above and below the first image. Taking multiple depths of images allowed for image correction since a portion of each image was out of focus and combined will give a full image.
4.3.2 Results
Figure 45 shows the variability of cell coverage in the corresponding well of each plate. Each image displays the Hoechst channel in grey-scale. Several samples were unable to be used, which resulted in only a reduction of the number of samples that were able to be imaged. Well loss was due to evaporation of the well’s liquid while in the refrigerator.
Figure 45. 8-Well Variability. Well 4 between all treatments A) Uncoated. B) 100 ug/ml & 5 min incubation. C) 100 ug/ml 30 min incubation D) 200 ug/ml 30 min incubation. Images taken in the middle
of the well where cells are the least populated.
Figure 46 depicts the variability of the Ethidium Homodimer-1 stain. Figure 46B was considered ideal where EthD-1 labels the dead cells as small localized and bright orbs. However, in addition to the dead cells, Figure 46A&C show disordered, non-symmetric and jagged objects that were remnants of detached cells that were killed by the UVB.
Figure 46. Ethidium Homodimer-1 Variability Between Wells. Same cell culture plate of 100 ug/ml 30 min incubation. A) Well 2, B) Well 3, C) Well 5.
4.3.3 Discussion
Figure 45 does show that there are more cells in the last image of the 200 ug/ml
concentration of PDL; however, application of the coating at this concentration was significantly harder to work with. The higher concentration made the PDL solution very viscous and would not spread out over the well, instead it balled up.
This experiment ended up being more about resolving experimental procedures than if the coating worked. It was found that when introducing UVB there was not a homogenous spread of cells between wells. Some wells looked as if they received more UVB exposure, like in Figure 46A well 2. All that was left were proteins that attached to the coating and were stained /with the EthD-1, coming up looking like debris and not individual orbs that we would hope to see in Figure 46B. Due to this variability a switch from 8-well plates to 1-well plates occurred for all following experiments.
Another major problem was the loss of wells due to evaporation, Figure 47. When the plates were stored in the refrigerator, the wells tended to dry out. Cell culture plates also tended to have the glass bottom crack because they were fragile. To remedy these problems the ‘Cell Culture Plate Holder’ was developed and can be found in Appendix A.1. The device was designed to hold the plates throughout the staining process so the bottom did not break. It also
had a lid with soft foam to gently apply pressure to the top of the culture plates to reduce evaporation when stored in the refrigerator.
Figure 47. Missing Wells. Representation of the four 8-well plates used. Darker wells signify being full of liquid, all others were missing liquid.
It was decided to use the 100 ug/ml concentration with an incubation time of 30 minutes. The higher concentration would not be used due to difficulty with its application to culture plates. The next experiment with PDL used 1-well plates to reduce potential UV
refraction from the walls. Seen in Figure 48 and detailed in Appendix A.1, the ‘Cell Culture Plate Holder’ was developed to reduce sample loss from cracking and evaporation, Figure 47. The p21 stain protocol that had been used before in the lab was introduced to the PDL and EthD-1 protocols as the final qualification step to observe any sort of adverse effects.
Figure 48. Cell Culture Plate Holder.