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.

We’ve also had some issues with actually seeing the cells on the microscope slides after fixing them with formaldehyde. Sometimes there are some cells, few and far between, but more often there are no cells at all, just dying cells and cell debris. For this reason, we are going to be more careful with our fixation protocol and periodically look at the cells under the microscope after treating the cells with formaldehyde and after the washes to see which step is either washing the cells off or killing the cells. Dr. Hinton consulted Dr. Allison for her opinion on what could be going wrong, and they think that we may be drying out the cells by completely aspirating all liquid from the cells and not moving quickly enough to replace the liquid covering the cells, thereby drying out the cells. Hopefully that’s the case, and we can fix this issue by the end of this experimental trial!

In more positive news, we’ve also started an experimental trial with N2A cells, a mouse neuroblastoma (brain cancer) cell line. It’ll be very interesting to compare these cells to our trials with PC12 cells once we have some slides next week!

Comments

  1. ammattei says:

    Hey! Thanks for giving us this update. PC12 cells as you well know can be quite finicky and fragile. Your perseverance in dealing with this important but far from user-friendly cell line is admirable. It is also exciting to read about your decision to incorporate a new cell line to further illuminate MK-STYX’s established and suspected roles in neurite induction and neuron morphology. Good luck with the rest of your summer work!

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