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.

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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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.

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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Adaptive Radiation and Stress Physiology of Vibrio Fischeri

So far this summer I have been steadily collecting and gathering data on the growth of V. fischeri smooth morphs and wrinkly spreaders on a salinity gradient. To do this I have been growing bacteria on LBS growth media with different concentrations of sodium chloride and measuring OD600 with a spectrophotometer to determine the population density that V. fischeri is able to grow to over a period of 24 hours .

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