So I have another question for you folks out there that have worked in primary neuronal cell culture. I'm wondering how you go about adding drugs and manipulating your neurons pharmacologically. The reason I ask is that I've been playing around with different ways of applying drugs, such as adding it to new media and then placing that on the cells, or using a solution of organic salts based on what I've seen in the literature. The issue I'm running into is that I cannot just add on fresh neurobasal media with the drugs because this sets off some signaling pathways (e.g, pERK, pCREB). I'm currently having an issue with adding a basic HEPES buffered salt solution because after about 30 minutes this results in a change in a different signaling pathway I'm interested in examining (but the pERK pathway seems to be mostly alright). At the moment I'm at a loss as to the best way to add drugs to my neurons without disrupting any pathways. Because of the experiments I plan on doing I would like to be able to completely remove the media and replace it with something else. I'm initially thinking perhaps the difference in sodium ion concentration in my HEPES buffered solution (it's 137 mM) and the Neurobasal (~75 mM) may be messing with my neurons when they are switched from neurobasal to the stimulation solution.
So my question is, what do you use to add drugs to your neurons? Do you just add it to the already present media? Do you use another solution? Do you notice any changes in various signaling pathways due to media/solution changes?
The next thing I'm going to try is using a solution that has an ion concentration that matches Neurobasal, however, I don't think I've ever seen something like this mentioned in the literature and I'm wondering if there is perhaps a reason why.
Thanks for reading, I hope someone out there has some insight!
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Neuronal Stimulation Protocols
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