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Blog Post #2

Status Update

Through my research, I am aiming to gather significant evidence to answer the question: Does exposure to carbaryl impact the insulin signaling pathway leading to insulin resistance in NIH-3T3 L1 cells?

Carbaryl is a chemical commonly used in pesticides for agricultural and residential use, but its effects on human health (particularly insulin disruption) are not well understood.

In the insulin signaling pathway, one of the last proteins to be phosphorylated is Akt; thus, the presence of phosphorylated-Akt (p-Akt) has been used in various studies as an indicator of successful insulin pathways.

By differentiating cells into adipocytes so they express insulin receptors, I hope to determine whether administering varying concentrations of carbaryl to cells affects the amount of p-Akt present, as a measure of whether carbaryl disrupts the insulin signaling pathway and interferes with glucose uptake.

In early February, I finally reached the milestone of successfully differentiating my L1 fibroblast cells into adipocytes! I had faced issues with overgrowth before, but due to planned timing, these cells survived and showed the visual morphology of adipocytes. Since this was successful, I was able to perform my control trials on these cells: they were starved using a media that didn’t contain nutrients to maximize stimulation effects, and then treated with a 100 nM concentration of insulin to trigger the insulin signaling pathway. After 20 minutes of this treatment, the cells were concentrated into pellets and frozen to preserve the amount of p-Akt in the cells.

I am doing well on continuing a healthy culture of cells; none of my undifferentiated plates have experienced infection or contamination. I’ve also grown a lot more comfortable working in the BSC due to regularly passaging cells, changing media, and other cell maintenance activities.

Challenges & Problem-Solving

One of the main issues I’ve faced has been, as mentioned before, overgrowth during the differentiation process. These cells multiply very fast, but my protocols require cells to be kept in each type of differentiation media for around two days. The first time I tried the differentiation process, I came back after a weekend when they should’ve been successfully differentiated to find out that they all died due to overgrowth.

After talking with one of my mentors, Andrew Neumann, we determined that it was due to overgrowth; I instead adapted to these challenges by seeding cells at a much lower starting density to ensure they had more time to grow. I also found that keeping the cells in certain media for shorter periods of time still works, as seen by the plate I was able to successfully differentiate.

Another challenge I’ve been grappling with is the fact that I won't know if my project is successful until I complete the Western Blot to analyze my data at Anschutz Medical Campus. It makes gathering data slightly discouraging, but to mitigate this I’ve worked on making my protocols as detailed as possible, informing the process with procedures that worked in other published research papers.

Data Highlight: A Successful vs Unsuccessful Differentiation Process

Attached are images of my first attempt at differentiating cells. 2L and 2M were the 6-well plates I first tried the differentiation process on, and all these images are taken from well 1 of that 2L. To help you get a better idea of what you're looking at, the darker, oval-shaped items in Picture 1 are undifferentiated NIH3T3-L1 fibroblast cells. On January 28th, they had been in Differentiation Media I for one day. On January 29th, Differentiation Media I was replaced by Differentiation Media II; however, as you can see, the cells are very closely concentrated together. This indicates they are at almost 95% confluence; cells being that close together means they start competing for space. That was proven on February 2nd, the Monday I came back and saw almost all the cells had died. The brown tissue-like clumps are dead, floating cells. I had expected cells to stop growing after being placed in Differentiation Media I, so I started the process when cells were already at 70% confluence. I learned from this process that I should instead start the differentiation process when cells are at a low confluence, around 40%.

I tried the process again. First, I seeded cells at a lower percentage than normal into the 6-well plates 3Y and 3Z; attached are images of 3Y. As you can see, after they had adhered to the plate on February 5th, the cells are much more spread out because they hadn't had a chance to multiply too much yet. The next day, on February 6th, the Basal Media I (the media cells are regularly cultured in) was replaced with Differentiation Media I. After the weekend, on February 9th, Differentiation Media I was replaced by Differentiation Media II; as you can see in the images of Well 4 and Well 2, they are more clustered than before, but cells are already displaying the morphology of adipocytes. Adipocytes appear brighter and rounder under the brightfield microscope compared to fibroblast cells. Finally, by February 11th, I had replaced Differentiation Media II with Basal Media II, a medium similar to regular Basal Media I but containing insulin to keep the adipocytes healthy. Having successfully differentiated 5 wells of 3Y and 4 wells of 3Z, I replaced Basal Media II with a starvation medium (containing no growth hormones or insulin) to ensure cells weren't already stimulated, then administered insulin the next day to trigger the insulin signaling pathway.

Reflection

Throughout this process, I’ve learned a lot both about the topic and myself as a researcher. I learned not only how to make different cell media and procedures, but also the purpose of different chemicals and how exactly they impact the behavior of cells. During working, I’ve become far more comfortable with procedures like making new media, passaging cells, plating them into 6-well plates, checking confluence, and more.

Additionally, when I started the project I had uncertainty about my own ability to keep going in case things weren’t going right. For instance, after the 6-well plates 2L and 2M died, I was very discouraged due to the amount of money that went into making the differentiation medias, the plates themselves, and more; however, that just taught me to be more resourceful in how I use the different cell materials in the lab, and I adjusted my working style to successfully differentiate 3Y and 3Z. I look forward to successfully differentiating more cells so I can complete my experimental trials!

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