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Overview of Methods
After learning cell culture protocols in pretrials and becoming more confident in my abilities, I entered Experimental Trials where I seeded the cells into two 96 well plates and exposed one plate to 1:5000 concentration of Roundup for 21 hours and 30 minutes. Both plates were stained with the CellROX assay so oxidative stress could be detected. The 96 well plates were placed into a microplate reader which provided me with the absorbance value of each well. Absorbance values correspond to the concentration of cells emitting a wavelength of 490 nanometers, signifying the occurrence of oxidative stress. I recorded the value of each well and calculated the mean and standard deviation for each plate. Using this data, I performed a 2 sample 2 tailed T test to calculate a P value of 0.00037. Because this value is lower than the significance level 0.01, I concluded that Roundup caused significantly more oxidative stress than the control.
Infected mold plate
Mold Infection
Mold Infection
I noticed a brown clump in the cell media bottle, signifying a fungal mold infection. I continued growing my plates anyway because the cells were doing well. However, after a few days, the infection spread onto the plates, and they became unusable for data collection. Here is a photograph of one of the infected plates. Because of the infection, I had to throw all of my plates away and restart with the second vial of cells my mentor donated me. I am unsure what the exact cause of the infection was, but it was clearly some type of lack of sterility. I have been trying to keep better sterile procedures which prevented future infections.
New Splitting Protocols
New Splitting Protocols
I plated my second vial of cells and began growing the plates. They were doing well, and I was splitting them without issues throughout the week. Unfortunately, after leaving them over the weekend, all of the cells died. I wasn't sure what the issue was, as I didn't have any previous problems with my splitting protocols. My mentor, Andrew Neumann, kindly came to the Rock Canyon lab to help assess my protocols. He found that I could have been using too much Trypsin, a chemical used in splitting that helps detach the adhering cells from the plate. Trypsin "eats" away at cells to help them detach and is ideally neutralized by adding media. However, using too large a ratio of Trypsin or leaving the cells in Trypsin with no media for too long can both cause the cells to be eaten away completely. Andrew helped me develop new protocols that used a smaller ratio of Trypsin. He also recommended manually detaching cells using a micropipette instead of adding more Trypsin if cells don't detach. I used these new methods to finish my project and found they worked much better. This change in methodology ultimately helped me continue my project and more confidently split plates.
Andrew watching me perform the new protocols to make sure I was proficient in them.
More Cell Death
More Cell Death
The cells were doing well and growing quickly. I hoped to split them one last time so I could have a total of 10 plates in order to have multiple plates of two different concentrations and a control. I was planning on seeding them into the 96 well plate after the weekend. However, on Monday I found that eight of the ten plates had died. I called Andrew because I wasn't sure what could have happened for some plates to survive and others die. Andrew did not know either, but suggested it could have been overconfluency in some of the plates, meaning the cells grew too much over the weekend and used up the resources.
I did my best to salvage the two plates that still had live cells and continued growing them. Because of this road bump, I could no longer have two different concentrations of Roundup. Instead, I would only have enough cells to seed one 96 well plate with cells exposed to a 1:5000 ratio of Roundup and one well plate with just growth media to serve as a control.
96 Well Plate Seeding
96 Well Plate Seeding
When seeding cells into the 96 well plate, I followed similar splitting protocols except instead of splitting into a new petri plate, I split the cells into 96 wells. Cells should be left to adhere to the wells for 24-48 hours. However, due to time constraints, I let them adhere over the weekend which resulted in 72 hours. By this time, they were overconfluent, and cells were clumping together. However, the cells were not dead, so I decided to use the plates to continue experimental trials anyway, as I didn't have enough time to restart again. This was a minor limitation to my project.
Using a micropipette to seed cells into 96 wells.
96 well plate, inner 60 wells were used because outer wells must be left empty to avoid evaporation
Adding CellROX reagent to wells
Microplate reader and computer set up for data analysis
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