Use serum free medium. See Original publication of Romijn et al., 1984 Neurosci Biobehav Rev 8:301-334, now available with companies like Sigma etc. See also publications of T.Heida et al., IEEE Trans Biomed Eng 48: 921-930 and J.Neurosci meth 110:37-44.. Also look into Buitenweg J all results based on reduced glia by serum free medium. You need not to get all glia suppressed, since on a single glia layer neurons will function better. Underground is very important see publications Ruardij.

Good luck and success. Tell you a small story: we had for 3 month no effective cultures (early days of neuron culturing in the 1980ies), and found out that yeast was the cause. Later on it came out that one of the girls working with our cultures lived next to an industry that used solely yeast, brought in the air, found it in her hair. So don't be desperate, you Always find a solution.

Ara-C is meant to be some peoples choice of glial cell proliferation inhibitor. I guess if you only put it in after 3-5 days after plating then maybe you can limit the astrocytes so its not one coherent layer - meaning hopefully more neurons close to the coverslip for tirf.

In addition to using serum free medium you can also add AraC into the culture medium from the third culture day on. AraC will inhibit astrocyte and fibroblast proliferation so that their levels stays very low and under this conditions calcium imaging is normally fine. Combining serum free medium and AraC normally leads to > 98% pure neuronal cultures.

In case cultures of single cells do not work out you could work with slice cultures. In slice cultures cells are still in their "normal" environment and you do not get overgrowth with glial cells.

Hello!

I only have experience with growing hippocampal cultures and the most dramatic difference on the glial growth is the age of the embryos you use for culture. There is always a big difference between e.g. E18-P1 and E16 cultures (mice). E16 cultures have very little glia present and you dont need to supplement the media with Ara-C- which inhibits growth of dividing cells (but also it can activate e.g. NF-kB responses so probably should be avoided). Whereas, if you are using tissue from postnatal animals, no matter what you do the glia will simply grow very effectively over the whole surface of your dish. Culture of rat hippcampus is a lot more straight forward and easier to get less glial cells even from P1 animals. I also found, like Enrico, that limiting serum will work, especially during the time of plating. When you leave the cells to adhere, you can just leave them for couple of hours with media supplemented with serum, the longer you live them in the presence of serum the more glial growth you will get. I hope this will help and good luck!

In my experience serum-free medium worked the best. I tried the ara-C, but it seemed toxic to the culture overall. What worked best for me was plating the cells in full medium with FBS, and replacing it with serum-free Neurobasal with B27 supplement 3-4 hours after plating (gentle and careful, but full medium replacement). Essentially the idea was to give cells enough time to attach, but do it still on the same day as dissection and plating. Much fewer astrocytes proliferate this way, and neurons were just fine in neurobasal.

Also, good coating of the glass is critical, my cells seemed really fussy about that. It if wasn't good enough, neurons tended to cluster around/on top of astrocytes. I used poly-L-lysine, and ended up making a dilution fresh each time and coating overnight (1-2 hrs wasn't long enough).

I cultured cortical neurons from E14 embryos. I'd also second the comment about the age of the embryo playing a role, you get more astrocyte proliferation at the end of pregnancy.

Hi Maiban,

I agree with the previous anwers, you can work in a serum-free medium. It is impossible to completely get rid of glial cells, particularly if you use 10-12 DIV cultures. I attach the protocol we use for cortical neuron-enriched cultures.

Good luck!

Valérie

I use E14 embryos. The age is very important to eliminate glia cells.

Grown in serumfree Neurobasal medium with B27 supplement.

Use FdU 5µM final concentration the day after setup.

Ara-C is toxic to neurons

With this procedure you will get less than 2-3 % glial cells in your culture

I am planning to start using serum free medium and follow the protocol that you'll have suggested for my next dissection that is coming in few days. I will post it here if I see a difference in my culture. Also, if using serum free medium don't give me cultures good enough for TIRF then I will have to start using embryonic animals. Looks like a combination of serum free media, embryonic animals and FDU or AraC is the best way to go. 

Thank you very much for the answers everyone. very good points. I really appreciate it.  That really helped.