I want to do a serial dilution of 10uM, 100uM to 1mM of glutamic acid on my primary neuronal cultures in order to look at gene expression level changes over time (after exposure to glutamic acid). I was wondering how long I can leave the glutamate in the medium without it causing glutamate toxicity. I know 1mM for sure will, but I was wondering about the lower levels. Is 1-2 hours enough? Or is it too much?
Thanks!
Diahann
Diahann: I don't have a specific answer for you, but thought I'd offer a few comments that might be helpful:
1. First ask yourself what kinds of gene expression you wish to study (i.e., it sounds as if you are not interested in any related to toxicity).
2. What kind of toxicity are you trying to avoid? (i.e., depending on the level of exposure, glutamate can cause rapid cell death, or a more delayed cell death)
3. Several other factors are likely to affect whether the exposure causes toxicity or not, including Ca++ concentration, glycine concentration (if NMDA-sensitive receptors are involved), temperature, and the presence of glia (which can greatly limit the amount of glutamate that reaches receptors).
I hope this is helpful. Good luck!
it would depend on a culture. If it is primary cortical/hippocampal cells they need to be 2 weeks old to fully express all glutamate receptors. If you didn't change them media to no glutamate on 2DIV you won't see any effect after glutamate. 10uM is not toxic (at least at my hand it is sublethal). 100-150uM will damaged them by 3-6h. And 1mM I've never tried.
Hi Diahann
According to the previous reply, it depend on your neuronal culture (cortical, hippocampal, pure or mixed with glial cells) and their maturity. Unfortunately I've no idea about glutamate concentration/time to use, but a short exposure to 50µM of NMDA (1h) induced a delayed neuronal death (after 24h). I think you should check your cell viability at different time (between 1 and 24h) for different concentrations of glutamate.
Good luck
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