Week Eight

Thankfully, I figured out early in the week what had been killing the cells I have been working with. I had run out of the plates I had been using for the cells, and assumed that a slightly different type of plate would be fine - they were same diameter as what I had been using and were kept in the same drawer. However, I did not realize that the new plates I decided to use were too different, and did not have any substance on them that allowed the cells to attach. It is likely that the cells were not even dying, but since they were not attaching it seemed as though they were dying, because usually that's how we determine that cells have completely died. Regardless, the grad student corrected my mistake and my cells have been growing fine since I switched back to the correct plates. When people tell you to not make any assumptions in science, listen. Even the smallest assumptions can cause large problems. In this case, I mostly lost time, so the possibility that I would have to come in over the weekend became a definite.

I started working with fibroblasts now, which are cells found in connective tissues. Our particular cells are not from an immortal cell line like the HEK 293s, so we have to avoid splitting them as they will stop growing properly after too many passages. It is exciting to have started my work with the fibroblasts since they are the next step in the determining how effective the treatment I am testing is. It is also quite interesting to work with a new cell type, since my work during this summer experience is the first time I have worked with cell cultures, and it is always fascinating to see cells under the microscope as they grow.

Figure 1. Extremely confluent human dermal fibroblasts; their function in connective
tissues apparent in their morphology and growth patterns (CELLnTEC).

Last week we had attempted to replicate certain plasmids that we had through a transformation with bacteria. In a transformation, the bacteria used take in the plasmid DNA and as the bacteria replicates - which it does quickly - the plasmid DNA replicates as well. After the bacteria has replicated enough, the plasmids can be isolated again from the bacteria and used for other purposes. We performed the isolation this week and the DNA concentrations were what we expected, showing that we had successful replicated the plasmids. One of the major concerns in a transformation is that only the bacteria that has taken in the plasmid is what should be replicating, otherwise it is impossible to differentiate which bacteria actually has the plasmid in them or not. The plasmid has an antibiotic resistant gene in it that is expressed after a short time once it has entered a bacterium, so the addition of antibiotics to the media in which the bacteria will grow is sufficient to kill any unwanted bacteria.

Figure 2. The transformation process for replicating plasmids (Addgene).

On Monday of this week, once I had resolved the issue with my cells, I was ready to work and decided to start the Western Blot I needed to complete. In our lab, Western Blotting takes two days to complete once protein samples have been collected, with the first day being the longest. Even though I started the Western Blot at noon, it really was too late since the entire protocol for the first day - with many waiting periods - takes about seven hours, which I hadn't fully realized until I had started. The lab technician I work with was going to warn me, but honestly it wouldn't have mattered much if she had - I was determined to start the Western Blot and I didn't mind staying late that much, especially considering the protocol is not one where you actively must be involved the whole time. The extended time just meant that I would go home later that day, but have my results sooner and have a calmer week overall past that point. One of the hardest parts of working long hours when you haven't anticipated it before you left for the lab is not having enough food for the time you are there. However, later in the day the lab tech and graduate student were still there with me. the graduate student had a moment to run out and went to get snacks - I thought for himself - and came back with a dozen donuts, which he offered to the lab tech and I. It was a small gesture but he really didn't have to bring us back food, and it made any doubt about my decision to stay late go away.

Figure 3. Mitosis illustrated with donuts (Van Alest, 2005) 

References:

Addgene. n.d. Bacterial Transformation. <https://www.addgene.org/plasmid-protocols/bacterial-transformation/>.

CELLnTEC. n.d. Primary Human Dermal Fibroblasts. <http://cellntec.com/products/skin/fibroblast-cells/>.

Van Alest, K. 2005. Cellular mitosis. Photograph. <http://www.kevinvanaelst.com/photo10.html>.