On Friday, I met with my mentor for the last time in 2013. We went over the data collected from my wound healing assay experiment last week. For the most part, my data was as expected. However, there were two wells that did not follow the general trend of my results. Possible error in these two wells could have been that I did not have the same amount of cells in these wells as the other six. Therefore, the graphs of these were significantly lower than the average. However, the cells in these wells caught up to the others after about 15 hours.
I also passaged my BSC-1 cells and MDCK cells. With practice, I am learning how to better passage these cells. The MDCK cells are the hardest to passage because they have tight junctions and are therefore difficult to separate from one another and the bottom of the flask. On Friday, I only used about one drop of cell culture in each of my new flasks because I will not be working with the cells for a few weeks. We do not want the cells to multiply and grow too quickly because they will use up all of the nutrients in the medium. Dr. Keese will be babysitting my cells while I am away, making sure they have enough medium and are healthy and pathogen-free!
Dr. Keese taught me how to manipulate the ECIS software to better understand my data. My favorite function saws the 3D graph option! He showed me how to put my graphs into three dimensions, and how to interpret these results. The 3D graphs plotted time (hours), frequency (hz) and impedance (Z). I would have loved to attach the graph of my results, but I had a bit of trouble getting the data onto my computer. I will try to get these images uploaded onto my blog for my next post. Sadly, I will have to wait until January 10th until my next visit to Applied Biophysics!
Tuesday, December 17, 2013
Monday, December 9, 2013
On Friday, I passaged my BSC-1 cells to make two new flasks of cells. I will be using these cells in a co-culture experient in which I will combine BSC-1 and MDCK cells together and examine their behavior with an ECIS run. I also passaged MDCK cells today. These cells behave very differently than BSC-1 cells becasue they form tight junctions and are tough to suspend. In order to get these cells off of the bottom of the flask, I needed to add three rinses of EDTA, which is a solution that chelates the cells (takes the magnesium and calcium ions off of the cells). I then added trypsin, which also helps suspend the cells. I then took my MDCK cells, added medium, and put 400 µL of this cell-medium mixture into seven wells of an 8W1E electrode. The eigth well is just filled with medium, no cells.
I then plugged my electrode array into the ECIS port, and started to run my experiment! In this experiment, I will be performing a wound healing assay. In a wound healing assay, the electrode sends out a current of electricity that will kill the cells in the vicinity of the electrode. In this experiment, I sent a 1400 uA current through four wells for 20 seconds.This process will cause the cell membranes to become porous, and medium will leak into the cell, causing it to die. Why would I want to kill of perfectly healthy cells? Because then we can see how the other cells will move in to heal the wound. We had to wait for the cells to settle to the bottom of the electrode and grow into a confluent layer before we wounded the cells. I set the wound time for 9 hours into the experiment, and I will see what my results look like this Friday!
I was able to connect some of my knowledge about genetic modification that I had learned in AP Biology to this wounding process. ECIS allows one to either wound their cells, completely killing the cells around the electrode, or electroporate them. Electroporating cells introduces a high frequency current that damages cell wall (not killing them), causing them to become more porous. The increased porosity of the cells allows substances (such as large or non polar molecules) that could not usually pass through the selective cell membrane to enter. Electroporation allows a scientist to introduce genetic information into the cells, often by a virus, changing the cells' DNA.
I then plugged my electrode array into the ECIS port, and started to run my experiment! In this experiment, I will be performing a wound healing assay. In a wound healing assay, the electrode sends out a current of electricity that will kill the cells in the vicinity of the electrode. In this experiment, I sent a 1400 uA current through four wells for 20 seconds.This process will cause the cell membranes to become porous, and medium will leak into the cell, causing it to die. Why would I want to kill of perfectly healthy cells? Because then we can see how the other cells will move in to heal the wound. We had to wait for the cells to settle to the bottom of the electrode and grow into a confluent layer before we wounded the cells. I set the wound time for 9 hours into the experiment, and I will see what my results look like this Friday!
I was able to connect some of my knowledge about genetic modification that I had learned in AP Biology to this wounding process. ECIS allows one to either wound their cells, completely killing the cells around the electrode, or electroporate them. Electroporating cells introduces a high frequency current that damages cell wall (not killing them), causing them to become more porous. The increased porosity of the cells allows substances (such as large or non polar molecules) that could not usually pass through the selective cell membrane to enter. Electroporation allows a scientist to introduce genetic information into the cells, often by a virus, changing the cells' DNA.
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