Week 4- What the HEK?!

Anita M -

This week I plated HEK293 cells into two six-well plates. Each well has roughly 750,000 cells suspended in 2 mL of media (media provides cells nutrients).
Here’s a picture of my cells!

After plating, I incubated them overnight and added a GRASP55 plasmid and a control plasmid the next day. This plasmids are mixed with a reagent that coats the plasmids in a lipid layer, allowing them to pass through the plasma membrane of the cells. However, it is still a relatively large molecule, so prolonged exposure to the plasmid can cause damage to the cells. To combat this, we only expose the cells to the plasmids for 4-8 hours and then aspirate the old plasmid/media mixture off and put back in 2 mL of fresh media.
Attached is a diagram of a plasmid. The blue region contains our gene of interest, and ribosomes in the cell bind to the promoter region to begin making the GRASP55 protein for the cell.


Created in https://BioRender.com

Unfortunately, my cells were too confluent and when we removed the plasmid/media mixture, we noticed a large chunks of cells removed in some of the wells. We ended up having to toss the plates, since the number of cells in each well is no longer consistent and if we continued with the experiment, we would have unreliable data.
This is a minor setback, but it can easily be fixed by simply plating a smaller number of cells next time.

Attached is a picture of my “green sheet,” which is basically a guide I wrote that clearly outlines each step of the project. The goal is to plan out at much as possible, and keep modifying it as your project continues. Since this is a continuous four day procedure (we can freeze the cells after Day 4 and continue the project after a few days if needed), we will have to start at Day 0 on Monday of next week.

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    aashi_h
    Hi Anita, I was drawn in by the title of your post! I love the research you are doing. Why do you think that the cells were confluent when you removed the mixture? Why did this phenomenon occur?
    March 5, 2025 at 6:34 pm - Reply
    anita_m
    Hey Aashi! Thanks for your question! There are a couple reasons the cells became overconfluent. The first is that our calculations could have been off, and we actually plated more than 750,000 cells. For reference, 100% confluency is around 200,000 cells per well for the HEK line, so even adding a couple hundred thousand cells more can add up quickly as the cells divide. The other is that these cells ended up dividing quicker than expected. The general dividing time for HEK is 16 hours and we left these cells to incubate for a little over 24 hours. Ideally, we should have seen around 80% confluency. As you can see in the photo attached, it was closer to 90%. When we removed the mixture, the chunks of cells that detached from the plate is characteristic of cell death caused by over confluency.
    March 7, 2025 at 12:03 pm - Reply
    camille_bennett
    Hi Anita, great images. I think it is also great that you have created a guide for your project. How do you ensure that your protocol is adaptable to setbacks like the one you encountered? How does this guide help you stay organized in the lab?
    March 10, 2025 at 1:49 pm - Reply
    anita_m
    Hey Ms. Bennett, Great question! We try to keep our protocol adaptable by writing down the details of each step of the process (the concentration of substances, volumes of substances, etc.). If something in the experiment doesn't go according to the plan, we can go back to those details and see what could've went wrong/what we'd need to change. We also write in pencil, so we can change certain parts/details of the protocol if necessary. The guide helps us stay organized by ensuring that we plan ahead for our experiments and know exactly what each part of our experiment entails. It also helps other people repeat similar experiments in the future.
    March 14, 2025 at 10:04 am - Reply

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