Have you tried "panning" for the microglia first? See https://www.nature.com/articles/srep19371

As compared to shaking method for microglia isolation "mild trypsinization" method gives much better yield almost 40-50% of the seeding density and also microglia isolated from this method are considered to be in resting state.

https://www.ncbi.nlm.nih.gov/pubmed/14603460

Article Characteristics of primary rat microglia isolated from mixed...

Hello! First, I highly recommend you to use this medium (4.5 mg/mL glucose – 0.584 mg/mL L-glutamine (DMEM – Gibco), 10% FBS (Gibco - have to be a good serum), 1 mM sodium pyruvate (Lonza), 100 U/mL Penicillin-100 mg/mL Streptomycin). You can improve your yield adding M-CSF (need to be more than 10ng/ml, what can be expensive) in your mixed glial culture when it achieve 80-90% confluence. I highly recommend you to check the methods in this paper: Maternal inflammation induces immune activation of fetal microglia and leads to disrupted microglia immune responses, behavior, and learning performance in adulthood. 10.1016/j.nbd.2017.07.017. Good Luck!

Just like what was recommended before, I would recommend immunopanning for the microglia. There are some ways that you could definitely improve your yield according to the information given. You stated that you used 3 brains for 1 T75 flask, and our lab has found that to be way too much! We take one mouse pup brain (P0-P1) and split it into 2 different T75 flasks. I would highly recommend this publication, because you can get the highest yield per pup with up to 97% purity of microglia:

Article A Simple Magnetic Separation Method for High-Yield Isolation...

You may try this revised method as well!

I would not perform a second harvest, as the microglia from the second harvest are a lot different from the first. It is difficult to compare the results obtained from those two populations.

I agree with Clarissa's suggestion. M-CSF (10ng/ml) is very important for microglia proliferation. We did not find the many difference between the 1st and other harvest by using the marker labeling and patch clamp.

Hi Vikas,

Would the mild trypsinization method work though given that mouse microglia remain attached to astrocytes in mixed culture- unlike the rat microglia in the paper you reference which tend to float in the supernatant. It is probably why they were able to perform mild trypsinization right?

I was also debating using this technique in lieu of shaking because I am doing the same work Sailesh is doing, until I learned about this fact. What do you think?

Hello Rania,

I think mild trypsinization method for microglia isolation works well for both mouse and rat models. It is easier to perform and we obtain sufficient quantity of cells with excellent purity.

Hi Vikas,

Did you have to separate your microglia from your astrocytes post-mild trypsinization if it's being said that in mouse-derived cultures, and unlike rat-derived cultures, microglia remain attached to astrocytes in the first place in mixed glial cultures? I am curious to know if you had to do this as I do not expect mild trypsinization to be sensitive to microglia every time we try isolating them from the mixed glia.

Thanks,

Rania

Hi Rania,

After mild trypsinization of mix glial culture and adding fresh medium to adherent cells, we obtain more than 95 % cells are microglia from mouse pups. After mild trypsinization an upper layer of cells comes out containing majority of astrocytes while microglia remains adhered to culture plate. In recently published article (in journal of neuroinflammation indicated in previous answer) authors have isolated microglia from rats and showed higher yields of microglia from mild trypsinization method as compared to shaking method.

Another option would be performing a MACS separation. Trypsinize the mixed glia and use a combination of CD11b and CD45 beads. The positive fraction is microglia in high purity. This works very well in our hands.