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.

Odds, Ends, Protein Concentrations, and CAT Standards

CAT KILLER STRIKES AGAIN! My latest victim bore the additional burden of the lowest protein concentration of any transfected samples thus far. Could this be due to the room temperature lysis buffer incubation I had performed? I’m not sure. I would be able to narrow down possible causes of this low protein concentration had I done what was suggested (take a 10 microliter portion of one of the samples after the thirty-minute lysis buffer incubation and observe whether or not the cells looked whole). I forgot this step in a rush to determine protein yield with high hopes that this would be my first successful CAT ELISA with a high protein yield due to the room temperature lysis conditions. Since CAT ELISAs depend on cell lysis for effective measurement of protein concentrations, whole cells appearing after the incubation with lysis buffer would lower measured protein concentrations. Subsequent centrifugation of whole cell samples would cause high levels of protein loss as the protein would pellet out contained in the cell. Instead of wasting plasmids on a process that my or may not work, the next step is seeding normal HeLa cells in plates and subjecting different plates to room temperature, ice and half-room temperature/half-ice lysis to determine which conditions are least conducive to whole cell state maintenance. Another recent critical change to the protocol is the use of 10 microliters of thyroid hormone rather than the one microliter amounts previously used; if I ever proceed beyond the Bradford Assay again, this larger amount of thyroid hormone will hopefully solve the issue of no apparent thyroid hormone induction. Unfortunately, I’m also still getting exponential CAT Standard curves where they should be linear. I’m not sure how to fix this problem besides trying to epitomize vigorous pipetting to ensure uniform CAT Enzyme distribution.

[Read more…]