It’s all about Media, Media, Media

One has to admire the sheer amount of media required for tissue culture work. In the process of splitting cells, eight milliliters of fresh media is added to the old flask and only a small amount of this media, containing the cells that had reached a high confluency in the old flask, is added to a new flask containing around 15-20 milliliters of fresh media. Each plate in a CAT ELISA requires one milliliter of the resuspended cell mixture and nine milliliters of fresh media, if using a flask with proper confluency for seeding plates. The remainder of the resuspended cells in the fresh media and old flask are aspirated off, never to be used in a transfection or CAT ELISA. Each media change requires ten milliliters of fresh media to be added to each plate, so media changes alone require 120 milliliters of fresh media over the course of less than 24 hours when using six plates. I recently incubated some of the media I have been using for CAT ELISA transfections and found that nothing grew in it, suggesting that it’s not contaminated. This lack of contamination leaves the question of what is causing so many cells to detach from the plate surface post-transfection. It seems as though 70-80% of the cells present on the plate pre-transfection are being lost after tranfection and media changes. Transfection is a harsh process, however it seems unlikely that only 20-30% of the cells could survive. During my last transection, pre-warming the media seemed to help maintain higher confluency, but the vast majority of the cells did not survive the process. A low numberof surviving cells contributes to the low protein concentration issue so eliminating or mitigating the influence of this source of rampant cell death and detachment would be immensely helpful in the pursuit of elusive reasonable results. Considering the lack of time left in this summer research term, the next time I split a flask of cells, I plan to use the entire resuspension amount to seed two sets of plates and perform identical transfections for both. If the protein concentration from both transfection sets is high enough and the ELISA results are reasonable, I’ll be a little closer to CAT ELISAs with thyroid hormone receptor beta.