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.

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At Last, (Some) Success!

Over the past two weeks, I’ve been working to improve my protocols and technique, specifically transfection and fixation, since I had been having problems with seeing healthy cells expressing GFP on the microscope slides. I’d also had a few issues with cell confluency, getting low cell counts when using the hemocytometer to make the calculations for seeding new six-well plates. But as of a few days ago, it appears that we have mainly fixed our protocol issues! The slides we fixed on Monday have plenty of healthy cells, and we can see the expected expression of GFP as well. We were also having some problems with actually seeing neurites in the PC12 cells when they had been stimulated with NGF. After increasing the concentration of NGF for stimulation, however, this problem appears to have been solved as well since we’re seeing cells with neurites on the slides. This, along with speeding up the work pace and quickly replacing the liquid covering the cells during fixation to avoid drying the cells out, has finally resulted in our best-looking trial yet. Limiting the number of washes with formaldehyde and D-PBS has also appeared to help. There appear to be a lot of cells on the slides, so it’ll be interesting to score the slides and find out how the different DNA plasmids affected PC12 cell morphology and neurite formation.

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Fine-Tuning Our Experimental Protocols

Since my last blog post, a lot has happened. We ran a few more Western Blots, which were mostly successful. I have also been learning how to use the fluorescent microscope to examine and take pictures of the slides from our experimental trials, which unfortunately have not been very successful. Recently I’ve been having issues with cell confluency (how concentrated the cells are)– when the cells get too confluent, it provides a perfect breeding ground for fungus, as we found out a few weeks ago. When the cells aren’t confluent enough, however, we are forced to seed our six-well plates at lower than ideal concentrations. Low confluency has been the more prominent issue in recent weeks, requiring us to either seed at a lower concentration or simply wait for the cells to grow several more days.

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Protocols, Training, and First Western Blot

My first few weeks here on campus were full of learning procedures and protocols so that I could start doing experiments. The first week and a half, I began by learning how to make plasmids, which are circular strands of DNA that replicate independently. Plasmids can be used to manipulate cells to see what happens when you over- or under-express certain proteins. For example, in my lab’s most recently published paper (Banks et al 2017), we found that when MK-STYX is overexpressed in primary neurons, it changes their morphology. Plasmid preparation consists of transforming the target DNA into E. coli cells and then isolating the DNA from the bacteria after letting them multiply. I then determined the concentration of the plasmids using the Nanodrop.

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Abstract: Uncovering the Role of MK-STYX in Neuronal Development

In recent years, neurological diseases like Alzheimer’s, ALS, Parkinson’s, and dementia have become much more prevalent. Yet, the mechanism and development of such diseases largely remain enigmatic, and treatment is generally limited to supportive care. Neurodegenerative diseases generally involve a disruption in communication between neurons, whether by cell death or by fewer connections with other neurons (Gao and Hong 2008). My research centers on MK-STYX (mitogen-activated protein kinase phosphoserine/threonine/tyrosine-binding protein), a protein implicated in neuronal development. MK-STYX belongs to a group of proteins called pseudophosphatases, which lack catalytic activity but have homology to enzymes that dephosphorylate proteins, phosphatases (Hinton et al 2010). Though MK-STYX is catalytically inactive, it still plays a role in many cell signaling pathways, including cellular stress response, apoptosis, and neuronal development (Flowers et al 2014, Hinton et al 2010, Niemi et al 2011).

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