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┬áprotocol 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.

We’ve also had a better time with PC12 cell confluency as we’ve been more selective with which flasks are used to seed and more thorough with resuspending the cells in medium before calculating the cell concentration with the hemocytometer. As for the N2A cells we started experiments with recently, cell concentrations have been great (a little too high at first in fact), but we’re still trying to figure out the right molarity of retinoic acid (RA) we should use to stimulate the cells to form neurites. Soon, however, we should have a better idea of what concentration to use since we are testing different levels of RA on N2As to see which works the best. As the end of summer nears, I still have a lot of things to wrap up, but I’m excited to finally see some successful trials!

Comments

  1. Hi Kirstin! I am so glad to hear that your trials have finally started to work out. Given your last post, I am sure the frustration was building to a max before you finally figured out the most optimal protocol. Moving forward, what other cell lines will you be using to conduct your experiment?

  2. You are definitely not the only person struggling with fine-tuning your procedures, as I am also struggling to pay attention to the little details that would make the protocol work for my experiments as well. Despite summer almost coming to an end, I’m glad that you were able to see successful trials. Keep your energy up Kirstin, you will definitely see more successful trials soon!