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This is me pipetting cells from the 96 well plate into a centrifuge tube so trypan blue can be added
My Research:
E0771 cells are a spontaneous Triple Negative Breast Cancer (TNBC) derived from C57BL/6 mice (Le Naour et al., 2020). Making them a relevant model for cancer research for TNBC. TNBC is significantly more invasive than other cancer types and has the ability to quickly metastasize and spread throughout the bloodstream (National Breast Cancer, 2025). Once in the bloodstream, survival rates decrease to a 12% survival rate (American Cancer Society, 2025). The use of suspension flasks mimics TNBC once it has metastasized as the cells are free-floating in the media.
Onametostat is a PRMT5 inhibitor. PRMT5 aids in mRNA splicing, DNA replication, cell signaling, and gene expression, which can promote cancer cell growth and cancer progression (Li & Pützer, 2008). PRMT5 is overexpressed in TNBC, causing more rapid growth and proliferation (Wang et al., 2023). When acted upon by Onametatat, there is a decrease in methylation marks. This leads to changes in gene expression, improper splicing, and accumulation of faulty transcripts. In addition, there is increased DNA damage and less DNA repair, causing cell cycle arrest where the cell won't finish through the G1-S phase of mitosis (Hanard et al., 2018).
My Progress:
I have finished running all of my trials in the lab, and just finished all of my cell counts. This means all of my data is ready for me to run my statistical tests and interpret the data! My trials and the growing of the cells went much smoother than expected. Previously, contamination has been a big problem. However, my cells did not get a infection, and grew relatively quick. This helped my trials run much faster, giving me more time to actually count the cells.
This is me putting my cells into the hemocytometer so I can take a picture of them under the microscope
Challenges:
One major challenge that occured was the amount of lab work I had on trials days, and I was working completely alone. On days that I started trials, I had to plate my cells into the 96-well plate, putting 220μL into each of the 60 well individually. I then Had to pipette out 20μL, add 20μL of trypan blue, and pipette it into the hemocytometer, before taking a picture under the microscope. This had to be repeated for every single one of the 60 wells in just one trial. This ended up taking a very long time, especially since I was working by myself, there were so many steps that I had to work through. To overcome this, I prepped as many things as I could in advance and then came into school early so I could get a headstart. I set certain gaols by certain amounts of time to keep myself on track, such as I have to finish 10 samples in the next 20 minutes. In addition, whenever I could feel myself slowing down I would take a quick break to walk around, eat a snack, and drink some water so I could stay efficient.
I ran into a similar problem when counting all the cells in the pictures. There were 360 pictures of cells with up to 300 cells per photo that I had to count individually. I found myself getting very board of counting dots in a photo, and it was hard to stay motivated through it. In order to overcome this, I again set small, achievable goals. Even on days where I didnt have class, I would set aside 30-45 minutes a day to just count cells. This stretched it out over a longer amount of time and made it feel much less daunting.
My Data:
Now that I have finished my cell counts, I am able to start analyzing the data! So far there is a difference in the amount of cell death between the control and experimental group, before and after the exposer period. This is a picture of my raw data for trial 3. The purple boxes are the averages of the cell counts, which is what is going to be used for my analysis. It was counted how many DEAD cells were in each photo. Column 2 shows the data for the control group, before the 48-hour exposure period. Column 3 is the cell counts for the control group after the 748-hour exposure period. As seen by this data, there was a decrease in the number of dead cells before and after the exposer period, showing that the cells were still growing in the well plate, and not dying. The 4th column shows the difference in cell death from before to after. Column 6 shows the amount of dead cells for the experimental group before the 48-hour expouser period. The 7th column shows the amount of dead cells after the 48-hour exposure period, with exposure to the Onametostat. The 8th column shows the difference in cell death from before and after the exposure time when cells were exposed to Onametostat. As seen by the averages at the bottom, there was a difference in the cell death with exposure to an Onametostat dosage of 0.00025μL.
My other 3 trials showed comparable data which helps to prove that this was no fluke in data. The averages are listed below.
Trial 1. Control before 48-hours: 76.67 Control after 48-hours: 52.63 Difference: -24.03
Trial 1. Experimental before exposure: 75.77 Experimental after exposure: 101.67 Difference: 25.9
Trial 2. Control before 48-hours: 122.4 Control after 48-hours: 27.93 Difference: -94.47
Trial 2. Experimental before exposure: 120.43 Experimental after exposure: 148.1 Difference: 27.67
What I have learned:
This project has taught me a lot about what it truly means to do research. It is not always glamorous and fun, and sometimes you just have to put your head down and power through. While working in the lab was fun, and at sometimes very interesting and technical, there were also times were the lab became very tedious and time consuming. When working on a project this size, on my own, it was very difficult to get my data as I had to take my cell counts in all one sitting. In addition, the cell count process became very tedious especially by myself. I learned a lot about what it means to work by yourself, and the discipline it takes to complete a project of this size independently. I learned that I need small goals to work towards so that things seem more attainable and rewards when I reach those goals to keep myself going.
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