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.

Training Complete

For the past few weeks, Dr. Hinton has trained me and my fellow summer lab members on various procedures and experimental techniques that are commonly used in lab. We have learned how to properly use the tissue culture room, which is a sterile room where we are able to culture various cell lines. There, we are able to treat our cells with different plasmids, giving them different expressions, and under treatment, we can see how these different expressions react under treatments. Along with that we have learned how to make more plasmids when we are running low. This process can be extensive, especially when they are multiple plasmids that need to be replenished. We used E. Coli bacteria to help culture the plasmids. Once the bacteria was transformed with corresponding plasmids, we broke up the cytoskeleton and other extracellular membrane proteins  to isolate the plasmids for further experiments. We have also learned how to do Western blots. Western blots help detect certain proteins with the help of antibodies that are specific to the proteins of interest. Western blots are done by first running a 2D gel with an electric current. After that, the gel is placed into a machine to transfer the data onto a membrane which allows for ease with antibody tagging. Training has gone very well so far, and I cannot wait to start my independent project.

Localization of MK-STYX – Abstract

MK-STYX is a pseudophosphatase that has roles in neuronal cell development. MK-STYX is rendered catalytically inactive because of a lack of a specific amino acid sequence that is critical to having phosphatase function. While this may lead to the assumption that MK-STYX does not play a critical role in the human body, psuedophosphatases are actually a part of phosphorylation signaling cascades. I hope to look further into MK-STYX’s role in neurite development. Previous work into this showed that the presence of MK-STYX stimulates neurite formation. In addition, these growths also demonstrated axonal and dendritic properties. My project will be to look at where exactly MK-STYX is localized in the neuron, the cell body or the dendrites, and what other proteins it interacts with to cause neurite growth. The localization of a protein in a cell provides information on its function. For example, if MK-STYX is discovered to be localized in the dendrites of neurons, it suggests that MK-STYX has a role in neurons connecting to each other. This has applications into neurodegenerative diseases like Alzheimer’s, Lou Gehrig’s disease (ALS) and dementia. 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 are also caused by similar deteriorating brain function. MK-STYX has the potential to treat these diseases by forming those needed connections.