My lab is now far past when we should have microglia in our 24-well cultures. We have plenty of what we assume to be astrocytes lining the bottoms of the entire wells but we cannot seem to get microglia to start growing atop the astrocytes. Any recommendations?
- We are using GlutaMAX DMEM with 10% FBS and 1% P/S, washing the wells once every two days with 1X HBSS and replacing the media with fresh media...
- We are now over 20 preps in and we cannot successfully obtain LPS-induced nitrite concentrations from our cultures because it seems that our microglia are not growing...
-Has anybody had this problem before?
I obtained nitrite from BV-2 cells, while I could not see nitrite from primary mouse microglia upon LPS treatment. (Nitrite oxide kit, Griess Reagent System from Promega)
I don't know precisely why primary mouse microglia can not produce nitric oxide. Still, you may see that numerous papers did not show nitric oxide results from LPS-induced primary mouse microglia.
Astrocytes help to several things to collect high yield microglia.
(1) Proliferation of microglia relies on several factors, such as CSF-1 and TGF-beta, which components are produced from astrocytes. - See below for reference
(2) Microglia strongly attach culture vessels. However, the astrocyte layer covers the culture vessel, which prevents the attachment of microglia with the culture vessel. Finally, this astrocyte layer helps to collect microglia by tapping.
[See this article]
Bohlen, C. J., Bennett, F. C., Tucker, A. F., Collins, H. Y., Mulinyawe, S. B., & Barres, B. A. (2017). Diverse requirements for microglial survival, specification, and function revealed by defined-medium cultures. Neuron, 94(4), 759-773.
To get a high microglia density, it's important to collect your cells from mouse cortical brain from postnatal day 1-3. Your flasks should be coated with Poly-L-lysine. Two-three days after the initial seeding, remove the premium medium and add fresh warm DMEM/F12 with 10% FBS based culture medium to promote microglia rather than astrocytes growing. After this initial fresh medium changed, remove the existing DMEM/F12 via a serological pipette into a 50 mL centrifuge tube followed by an immediate replacement with half volume of fresh DMEM/F12 containing 10% FBS. Centrifuge the "old" medium " at 1,400 rpm (190 xg) to remove dead cells. Add half volume of the centrifuged supernatant to make the 20 mL required for the flask. This process can be repeated for up to 12-14 days. Once the mixed glia culture achieves a 90% confluence level, microglia cells from mixed glia culture will be ready for isolation (when overlaying microglia sits exposed on the astrocyte layer). Separate microglia from astrocytes by shaking the T75 flask at 180 rpm for 3-5 hours or 240 rpm for 1.5-2 hours on an orbital shaker placed into a humidified 37°C incubator with 5% CO2. Good luck.
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