I want to culture rat neuronal cells but have not done it before. I am planning on using neurobasalA media supplemented with B27 and l-glutamine. After isolating the neurons with papain and several trituation and centrifugation steps, will this media formulation keep the growth of glial cells to a minimum?
Hi Charles,
As Pete already mentioned above the best starting point is to perform a clean careful dissection of the tissue, but for those daredevil glia that manage to escape cytosine arabinoside (AraC) is the most commonly used anti mitotic drug (at least for hippocampal/cortical cultures). Another option is Myitomisyn C.
for more info on the mechanism
http://connection.ebscohost.com/c/articles/96949193/chromosome-breakage-induced-by-genotoxic-agents-mitomycin-c-cytosine-arabinoside-concentration-p53-dependent
Hey,
have you already consider to deplete the glia cells in your culture? There should be some reagents you could use at sigma, invitrogen millipore etc.
best regards
Hi Charles,
As Pete already mentioned above the best starting point is to perform a clean careful dissection of the tissue, but for those daredevil glia that manage to escape cytosine arabinoside (AraC) is the most commonly used anti mitotic drug (at least for hippocampal/cortical cultures). Another option is Myitomisyn C.
for more info on the mechanism
http://connection.ebscohost.com/c/articles/96949193/chromosome-breakage-induced-by-genotoxic-agents-mitomycin-c-cytosine-arabinoside-concentration-p53-dependent
FAC sorting for neurons is not helpful, since it will break off the axons when you insert the cells through the FACs needle. People commonly use AraC - however, if you are looking at any axon transport, it affects the rates and can commonly affect TRK receptor uptake of neurotrophins. I would suggest using N2 supplement instead of B27 with the L-glutamine in the neurobasal.
This paper shows how to remove glia from DRG cultures using a one time hit of Fudr and AraC
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0060558
Best of luck
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0060558
You can try to add Glycyl-Histidyl-Lysine (so called peptide of PICKART and THAYER) which was shown by SENSENBENNER et al to inhibit glial celle proliferation and increase survival time of neurons.
Ara C 5µM gives good and fairly reproducible results when studying glutamate toxicity
Hey everyone, I'm using Ara-C in mouse primary cortical neuron to inhibit glia cell growth, I wonder how important the timing for adding Ara-C is, the protocol at our lab is 24h after cell plating, however I've tried adding it in the culture medium during plating, and suggestions for different times are also available online. Ara-C seems to function by inhibiting DNA synthesis, does the timing have anything to do with its mechanisms? I'd like to hear your ideas, thanks!
Linn
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