Hi everyone, I'm isolating microglia from P1-2 mice and seeding microglia cells from 3 pups brains/T-25 flask. After seeding, I let this astrocytes-microglia mix culture grow for approx 2-3 weeks, and so far all good - astrocytes form a nice confluent layer with microglia growing on top. When I want to harvest pure microglia, I bang the flasks 2-3 times and collect all the medium containing microglia and plate it on 5cm bacteriological plates, 2 per each flask. I maintain these cells in DMEM low glucose with 10% FBS, 2% pen/strep/ampB, 2% L-glutamine/10% MCSF. What I observe is that cells do proliferate, and in 10-15 days they populate the plate. But they do not look like a primary microglia culture should look like: they maintain a very round and amoeboid shape at the beginning, and then become granular and unhealthy. Any idea about what could be wrong? Thank you in advance.
Hi Roberta,
please have a look in my paper here where we described the protocol in details:
https://www.frontiersin.org/articles/10.3389/fimmu.2020.546415/full
I think the problem is that microglia need glucose and DMEM low glucose could affect them a lot. Please try it with DMEM high glucose (follow my protocol and it will work).
Best,
Trim
See if this paper can be of help:
Pinheiro LP, Linden R, Mariante RM. Activation and function of murine primary microglia in the absence of the prion protein. J Neuroimmunol. 2015 Sep 15;286:25-32.
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