End of an Era

Today is my last day lab, and this last week has been packed. We finished up several trials, one of which we’ll actually be able to score for data since the cells look amazing. We also figured out the concentration of retinoic acid that we want to use for N2As since we were having some issues with actually seeing neurites and cell differentiation. When we fixed the cells, we didn’t see neurites and the cells looking very unhealthy. We realized that this was likely due to serum starving the cells the entire time we stimulated them (which was what was suggested to us by the provider of the new cell line) since we had been adding the RA to RPMI medium with 0.5% FBS instead of the normal 5% FBS, 10% HS. We made up a sample six-well plate of different concentrations of RA with normal RPMI after serum starving the cells overnight, and after 24 hours we were already starting to see neurites in each of the wells and the cells looked healthy. We found that the concentration of RA we had already been using was most effective, we just needed to stop serum starving for such a long period of time. Our future trials of N2As are sure to look much better.

We also actually started working with a new cell type as well, one that I’m going to be working with a lot in the future since my project’s question hinges on it– primary neurons. We specifically use hippocampal rat neurons. These cells are a bit difficult to work with since unlike PC12s and N2As, they don’t multiply. For this reason, we need to start an experiment with them as soon as we get the shipment from the company we order cells from to avoid having to actually harvest the cells ourselves by dissecting rat brains. The protocol for primary neurons is different since we immediately add the plasmids instead of letting newly seeded cells grow overnight, and we use electroporation to transfect the cells with DNA instead of lipofectamine. However, it will certainly be interesting to see how my short hairpin MK-STYX plasmids affect the primary neurons! At last, I’m able to start addressing the main question for my project– which domain of MK-STYX actually induces neurite formation.

Over the past 9 weeks, there have been a lot of ups and downs. I’ve dealt with contamination, low cell counts, no cells on the slides, cells drying out, and much more. But I’ve learned so much in these past two months, optimizing protocols, improving my technique, and finally getting a successful trial. At the beginning of the summer, I couldn’t even imagine being independent in the lab, and though I certainly still always have a lot of questions for Dr. Hinton and my fellow lab members, I’ve become much more comfortable working with different types of cells and working in tissue culture by myself. Research is definitely a slow-paced endeavor– you don’t get results for a manuscript in just a few weeks, you have to figure out how to deal with certain cell types and how to optimize protocols for what you are trying to do– but it’s a worthwhile one. I look forward to continuing my project this fall when I get back on campus!