As I have been writing this blog and reading the comments, I have realized that my explanations have been lagging in some areas. I apologize for that. This post is dedicated to filling all of the gaps (or as many as I can) and answering all of the questions that have been asked.
The first question takes us all the way back to my first post. In this post I discussed two experiments for which I had completed he analysis but they were no longer viable. Since I never explained why, so I'll do that now. The experiment was no longer viable because the 0Gy plate for the clonogenic assay did not have any cells.
The second question was about training; the most exciting/ unexpected part of training and procedures that surprised me. The most exciting, or maybe best is a better word, is that you understand the why things are done a certain way, the labels on everything, and how to not retract a disease better because of training. The most unexpected part is that they had to tell us that we should not pipette with our mouths. This means do not suck the liquid (that has biohazardous cells/substances in it) through your mouth and put it in the place you want it to go. To me, at least, it seemed a fairly straightforward concept, but after reading the training further I realized why. Biosaftey and preventing lab acquired diseases, is a fairly recent phenomena. I do not remember the exact date, but it was within the 20th century that biosafety began to be addressed.
The third question was: what did I learn about the process of freezing cells by watching Dr. Lacombe? First and foremost, I learned about the full procedure. It is fairly quick and simple, but you have to act fast, which is the second thing I learned. When working with cells it is all about acting fast and making sure they are in the right conditions. To freeze cells, it is ideal to have a device that can bring the cells down to -80 degrees Celsius at a constant rate before placing them in nitrogen gas. Since the lab does not have such a device, they place the cells in a freezer set to -80 degrees Celsius for 2 hours and then place them in nitrogen gas.
The final question was: what it means to lyse a sample of cells and analyze the proteins. To lyse a cell is to breakdown the membrane of a cell. Once the membrane has been broken down, the proteins within the cell have been released. Since this is associated with the cartridge I do not have a very deatials explanation for how they will be analyzing the proteins, but generally when analyzing proteins you check for the amount of specific proteins expressed within the cell.
That is all. I apologize to all anyone who feels the gaps have not been filled. If you have further questions, please ask and I will do my best to answer them.
For the freezing of the cells: Are you trying to lyse them when they are frozen, or is that to preserve them? In my lab, when we freeze cells to save them wee have to put them in a 12.5% glycerol solution to help guard them from ice crystals, so I can't tell if you are attempting to somehow lyse them in a constant manner or not.
ReplyDeleteI'm sorry about the confusion. We are freezing the cells to preserve them. They are frozen in 1 mL of a cryopreservation solutnion, which is a misture of the complete medium and 5% DMSO.DMSO, or Dimethyl sulfoxide, iss added to the cell media to reduce ice formation and cell death durign freezing.
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