I have isolated primary mixed glia from P0-P2 mouse cortices and use trypsin for dissociation. I am trying to expand my cultures for qPCR experiments. So far I have not had luck with my cell population growing. The cells attach and spread at various densities but when I perform a cell count during passaging, I am decreasing in overall cell number. I have tried various seeding densities (from 5,000 cells per cm2 to 100,000 cells per cm2). I am culturing in DMEM/F12 + 10% FBS. Has anyone had any issues scaling up primary mixed glia or have any tips? Do I need any other supplements to my media?
Hi Kossondra, I think you should reassess media environment conditions such as temperature, moisture , etc... your media supplement seems to be sufficient.
Have you tried stimulating with 2-mercaptoethanol? 50uM I think is typical.
Hi Kassondra,
I just used trypsin when I wanted to isolate microglia. For a mixed glial culture I used the following protocol which worked for me.
> remove meninges
> homogenize tissue with a 10 ml pipette, slowly triturate 30-40 times
> seed cells (1.5-2 brains per 75cm2 flask, 3 brains per 150 cm2 flask)
> leave cultures for 1 week without touching the bottles in DMEM+P/S+Glutamax+20%FCS
>after 1 week change media to DMEM but with 10% FCS
> after 5-7 days (12-14 days in culture) cells are separated by shaking (shake vigorously by hand)
> collect medium containing microglia and oligodendrocyte precursors (adherent cells are 98% astrocytes and ca. 2% microglia)
> seed cells for 30 -40 min on non-coated dishes (adherent cells are 99% microglia)
> remove medium containing oligodendrocytes, pellet cells (90-95% Oligos, 1% microglia. 5-10% astrocytes)
That was the original protocol: Article The dynamics of the LPS triggered inflammatory response of m...
Hope that helps,
Anja
Thanks Anja, what is the purpose of using glutamax for the first week only in culture?
The other components are of course in the medium as well. I just specified the FCS concentration because that is the only thing which has to be changed.
Wanted to follow up and say I fixed the issue by using PLL coated flasks or Sarstedt Cell+ flasks.
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