Hi Edward, I have very similar yields, see attached.

The differences between my and your protocols are as follows:

• 2 brains per PLL-coated flask;
• media: RPMI + 10% FBS + 5% pen/strep + 5% glutamine;
• shaking: after cca 8-10 days in vitro (when the astrocytes are confluent) - 200 rpm @ 37 °C for 4-6 hours, closed ventilation caps.

Could you send me that paper where you have found that much higher yields to compare it?

Check the type of pen/strep and note the serum.

Which type of pen/strep is the best to use?? and should we add horse serum on top of 10% FBS?

Hi Edward,

I used to work with primary microglia from newborn mice and remember that we almost always had yields of 0.5 - 1 mio cells per 75 cm2 flask. Essentially we followed this methodology:

- 2 brains (P0-2) per PLL-coated flask (IMPORTANT: remove meninges completely!)

- digestion with trypsin/DNase followed by mechanical dissociation

- culture in DMEM / 10% FCS / pen/strep with wash and fresh medium after 24h

- after formation of astrocytic monolayer (after 4-6 days) supplementation with conditioned medium (containing M-CSF)

- harvest of cells after another 3-4 days

So, the major difference in our protocol is the addition of the supplemented medium, which stimulates microglial proliferation. This conditioned medium is produced by fibroblasts and of course has quite a complex composition. However, the major factor driving microglial proliferation is M-CSF (CSF-1), which is highly abundant in the fibroblast culture supernatant. You can achieve a very similar effect by simply adding recombinant M-CSF to the mixed glial culture, however, this will be a very expensive approach compared to the conditioned medium.

If you are interested in the details of this protocol please check this paper:

CD14 and TRIF govern distinct responsiveness and responses in mouse microglial TLR4 challenges by structural variants of LPS (Brain Behav Immun. 2011 Jul;25(5):957-70). Or, alternatively, contact me if you have specific questions.

Best,

Tommy

Thanks everyone for responding!

To Tomas:  Here is a link to the protocol I found when I was searching about microglial yield:  http://www.bio-protocol.org/wenzhang.aspx?id=314  They claim that they can get upwards of 1,000,000 microglia / pup.

To Veronika:  Do you happen to know how the addition of horse serum helps increase the yield?  You also don't change the media at all after the harvest?

To Tommy:  I will send you a message directly.

Ted

Hi there,

I'm a bit late to the party, but here's a paper from our lab that we routinely employ to obtain primary microglia http://www.ncbi.nlm.nih.gov/pubmed/21074565.

Yields are upwards of 2 million per pup once you get the hang of the protocol.

Lemme know if you have any additional queries.

-Nikhil

Hi Edward,

the NTDK-P is the best choice for mouse brain tissue. It usually gives much better yields than a collagenase/Percoll protocol (even two -four times as many cells). It is important to use the exact buffer indicated in the data sheet, otherwise the perfomance will suffer. Have you tried CD11b (Microglia) MicroBeads for the isoaltion of primary microglia? It takes only 1 h and therefore is much faster than the almost two weeks of shake-off culture (which can always get contaminated of course). Another major advantage of the acute isolation is that you get the real cells. During two weeks of shake-off culture the cells can modify (this has even been shown on gene expression level for astrocytes isolated the same way). So the fast acute isolation is better for and the cells.

The CD11b (Microglia) MicroBeads even work very nicely for adult brain tissue if you use Myelin Removal Beads II to get rid of the myelin debris in the single cell suspension.

For all these products, instruments are available which can increase the level of standardization significantly and facilitate the procedure for upscaling. 

Please let us know in case you have more questions or need support in  your lab.

Best,

Sandra