Week 5- Oops…I did it again
Anita M -
The original plan for this week was to repeat the experiment from last week. So we started to do that, but on the second step (transfecting the HEK cells with the GRASP55 plasmid), we experienced the same phenomenon as last time. Cells were peeling off the plate and dying in clumps. We originally hypothesized that this was happening because the cells were too confluent. However, we only put 500,000 cells in each well this time and experienced the same result.
This prompted us to take a deeper look into our protocol and we determined that the cells were being left without media (a solution that contains nutrients for the cells) for too long during plasmid transfection. To combat this, we are going to make some technical modifications to our process. Unfortunately though, this does mean we have to restart the experiment next week.
Attached are some pictures of my cells pre-transfection (top) and post-transfection (bottom). As you can see the cells in the image on top are not as “crowded” as the picture of cells in my previous post, meaning these living cells were much less confluent, which is a good sign. This means we can continue to plate around 500,000 cells per well.
Furthermore, the bottom image shows the cells clumped together and “shriveled,” which is how we can tell that the cells are dead. The cells in the bottom image are a bit difficult to see, so I encourage turning up your brightness to get a better view.
This again is quite an unfortunate series of events, but research is all about figuring out how to keep moving forward despite encountering obstacles, so we’ll just try again!
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