Week 1 and 2

These past few weeks we have trained the new members of our labs. They have gone through plasmid training and tissue culture training. While the new members continued their training, we started our trials for the summer. We got new plasmids to work with that have more exact cuts at the domains of MK-STYX which is very exciting. Next week I will continue optimization trials.

Domain Specificity in N2A Cells

MK-STYX is known to play a role in neurite development. Additional neurite outgrowths can aid in forming better and more connections between neurons. When patients have Alzheimer’s, neurons are not able to reach other neurons due to the lack of connections between them, causing problems with memory and other mental functions. Other neurodegenerative disorders like Lou Gehrig’s disease (ALS) and dementia also are caused by similar deteriorating brain function. MK-STYX has the potential to treat these diseases by forming the needed connections. However, the specifics of how MK-STYX influences these pathways is still unclear. Previous work shows that the presence of MK-STYX in cells stimulates neurite formation. In addition, these growths also demonstrated axonal and dendritic properties. These previous experiments were conducted on PC-12 cells harvested from the adrenal medulla, a gland above the kidney, in rats.  Rat PC-12 cells are a well-established model for neurons. However, our lab is now interested in conducting experiments with N2A cells, a mouse neuroblastoma cell line. N2A cells, since they are neuronal cells, will serve as a better model in further experimentation while we investigate the specifics of MK-STYX function. My project will be to determine whether MK-STYX has a similar effect on N2A cells that it does on PC12 cells. N2A cells also display axonal growth, which relay messages to the brain. Observing these will allow us to better understand MK-STYX’s role in neuronal growth.

End of Summer

This last week has been surprisingly busy. There were a lot of loose ends to tie up before we left for 3 weeks. We finished another round of plasmid preparation so we have enough supplies to last us through the Fall semester. Plasmid preparation can be tedious so getting a head start on that will make our lives easier in the coming months. We also had to freeze down our cell lines. Since we won’t be here for the next weeks, we need to ensure our cell lines will be taken care of. Instead of replacing medium and flasks, we will freeze the cells down in nitrogen to keep them viable for experiments. We have also taken care of various lab maintenance issues like ordering more equipment to restock the lab. I also took the time to score a previous successful trial. Results were interesting, and I definitely was surprised at how much time it took me. Overall it has been a relatively smooth week in lab. I look forward to continuing my research in the Fall.

When Something Finally Worked

It happened! Our trial finally worked! After a few weeks straight of straight troubleshooting, we have finally managed to pull off a successful trial! Over the past few weeks we have dealt with problems with cell count. Before plating, we have to use a hemocytometer to count cells so that we can control how many cells we put into each well. When we first started conducting the trials on our own, our counts were so low, making it nearly impossible to start trials. We started to become more careful when culturing flasks and becoming more selective with which flasks to use. We also encountered issues with the fixation process throughout our trials rendering all of our cells dead on our slides. This was a problem that left us at a loss because we would see cells on our slides before we started fixation, but not afterwards. We figured out that when we were aspirating the washes, we were leaving our cells in too dry of an environment, so we began to increase our speed when fixing our cells. We also decreased the amount of washes so that we were not disturbing our cells too much. After our fixation and mounting process, we looked at our cells underneath a microscope and for our previous trials, there were very little neurites on the cells. This was concerning because we stimulated our cells with the proper neural growth factors that should cause more neurite extensions to grow. We ended up increasing the concentration of neural growth factor to help stimulate the growth. After all these changes made to our procedure, we have finally seen a successful trial! The cells look beautiful, healthy, and happy underneath he microscope with plenty of neurite growth. It has been very exciting to see the fruits of our labor finally come into fruition. I look forward to using these experimental techniques on other cell lines.

Experimental Troubleshooting

Our research project has been stalled because we have had multiple complications with our cell lines. First, one of our plates in which the cells were contained in grew some type of fungus while incubating. We had no idea what to do with it but unfortunately we had to toss that plate out. When we tried to restart the cell line, we tried to count the cells to get a higher concentration in the plates, but unfortunately the cells had not grown enough for us to seed them at a high concentration. These problems were interesting because we had no idea what was causing them. However, we have since continued our experiments by restarting the contaminated plates and seeding at lower concentrations. After we have completed the experiments, we look at the slides under the microscope to observe the fixed cells. Some of our slides have turned out well, but other slides have shown no cells at all. We are currently trying to troubleshoot our procedure and trying different ways to optimize it for the cells. We also realized that we had not been incubating our cells with nerve growth factor (NGF) for enough time and that might have been an issue with with not seeing neurites.