Transfection Updates
Bhuvi M -
Hello everyone! This week, I will discuss our transfection data results thus far.
Three days following the transfection, we changed the media of the cells. This was the same serum-free media. Here is a picture of the parental cell line with the transfected Axl from day 3.
On day 5, we noticed that some cells in the wells were lifting due to high confluency in the wells. I repeated each well. I used 1 mL of trypsin for the no-treatment wells to detach the cells from the well and incubated them for 10 minutes at 37 degrees Celsius. I, then, used 2mL of the serum-free media to neutralize the trypsin and put the mixture into 15mL tubes. I centrifuged these tubes at 1500 rpm for 5 minutes. This created a pellet of cells at the bottom, allowing me to aspirate the media above and resuspend the cells in fresh media. I then added the fresh media with cells to 60 mm plates and stored them in the incubator.
For the the transfected cells, we noticed that half the cells had lifted, while the other half remained attached to the well. To account for this, I resuspended the lifted cells and added fresh media to those attached.
In addition to replating and feeding the cells, I added the antibiotic to select the cells that had not been transfected. This antibiotic was added to the transfected wells and the kill plate. The expectation from adding the antibiotic is that all the cells in the kill plate will die because nothing was transfected into the cells, while a little more than half the cells will die in the trasfected wells since they did not uptake the new gene. G418 stops cells from making proteins, but if cells have the Axl gene, they can survive because they make neomycin phosphotransferase II which inactivates G418. Here are some pictures from day 10.
Thank you for reading! Hopefully, we will have enough cells next week to conduct a gene expression assay.