Hello,

I have had the same problem with my microglia cultures during a certain time. Unfortunately, I don't know exactly what the debris are. However, I know that for microglia isolation the serum is very important. When I had these troubles I changed my serum for 10% of FBS from Hyclone. Moreover, before plating the cells into Flasks (after the brain dissociation) I passe them through a 30um pre-separation filter (Miltenyi Biotec). Since I do that I don't have any trouble with debris in my cultures anymore. Also for cell isolation from the mixed glial culture I shake the cells at 220 rpm for 30 minutes and if I can still see some microglia on the astrocyte monolayer I briefly shake them by hand.

Hope it can help.

Shouldn't be too much cell debris really. As above, I use 10% FBS. I don't really see the problem if you do have debris, surely you can just plate down the microglia, and change the medium after they have plated down to get rid of the debris?

Dear Xianyuan,

If you really want to find out the identity of the round cells on top of your astrocyte monolayer, I suggest staining them to identify them.  From P2 animals, you might have oligodendrocytes and oligodendrocyte precursors in the mix.  And oddly enough, there were a few times that I did my dissociation SO gently that I actually still had neurons present!  This is mentioned in the Introduction of the Saura paper, as well as Fig. 1: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2140055/)

Since you seem more interested in the microglia that the astrocytes, I would think that your shaking protocol is probably fine, but you may want to look at the original 1986 Giulian & Baker paper in the Journal of Neuroscience for some additional hints: http://www.jneurosci.org/content/6/8/2163.long.

Having said that, I think that the speed you use might depend on the radius of action of the shaker that you use, so you may have to play with the speed a bit.  For example, I always wanted the astrocytes, and for my shaker I needed to shake 24 hours at 250 RPM, followed by two days of shaking at 100 RPM, to remove all of the small cells that had adhered to the astrocyte monolayer.  I would see debris in the supernatant media that was harvested after each shaking.  Since I also wanted oligodendrocytes, I just used the method suggested by Niels.  I would think that it would work for the harvested microglia as well.

Good Luck!

Jill

As others have stated, I can't say that debris has been all that evident in my microglia preps.  If the debris is of sufficient concern to you, you may consider passing your supernatant containing microglia thorough a filter of appropriate size to get rid of the debris.

In addition, there is a well established MACS protocol for removing microglia from MGC cultures using anti-CD11b, perhaps you may consider trying to perform the opposite type of selection, where you remove the astroglia from your microglia using an astroglia specific marker, such as GLAST.

Best.

Is the debris associated with the cells/culture (died in the flask; often large deposits at the flask walls), or is it associated with the serum?

Do you see the same debris you are referring to if you put serum into a culture plate? If the serum is the source of the debris, you could filter it/centrifuge it before use, or reduce the serum levels shortly before you collect cells.

I presume the debris is smaller than the cells, else you could try filtering/sieving to remove debris larger than the cells.

You could try reducing your centrifugation speed at the cell recovery step, to collect cells without too much debris accompanying it. Not an accurate intervention, but may help.

When plating, you could allow 30-40 mins for microglial adherence, then gently wash away some of the debris.

Thank you all for the helpful reply! I solve the problem now. I change the medium to DMEM + 1 % P/S + 10% FCS. Before plating I passed them through a 30um pre-separation filter. Change medium at the next day of plating. I could get around 500,000 cell per pup without major debris contamination. I have check them by flow cytometry. But by staining, they look ok, at least more than 90% is microglia marker positive cells.

By checking the mixed culture, I could see some dark round cells and yellow round cells on top of the astrocyte layer. According the paper shared by Jill (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2140055/), the dark cells are OPC and yellow cells are microglia. But after shaking (day10, 230rpm 2h), I feel more dark cells come off than the yellow cells. 

Does anyone tried reculture them after shaking and shake again in one week?

Typically, under phase contrast microscopy, OPCs are small, dark and slightly oval, while microglial are bright and round.

The link below shows a video of one of my mixed glial cultures with labels (from: doi:10.1016/j.nano.2014.07.010)

I usually shake microglia off (60-120 mins, >95% pure), then OPCs (overnight, 16-18 h, >95% pure, following differential adhesion step: put shaken media onto non-tissue culture-treated petri dish for 30 mins, allowing microglia to adhere, but not OPCs, then retrieve media with OPCs).

If left for ~1 week, OPCs and microglia will be replenished and can be shaken off in exactly the same way.

Article Development of a nanomaterial bio-screening platform for neu...

Imho, P1 pups give a slightly better yield of microglia compared to P2 (at least for C57Bl6) and so does 10% FBS with high glucose DMEM. I've also found that centrifugation after shaking off Microglia tends to activate them and they take longer to 'settle' down. Debris can be skipped by washing twice about an hour after plating and leaving them overnight before use. Re-culturing at least in my hands doesn't seem to produce the same quality of microglia as the first time culture so I usually do not re-culture (open to suggestions to improve this!) 

For microglia isolations I would strongly suggest you try the mild trypsinization method described by Saura (2003).  I have used this method routinely and the yield and purity generally exceeds that of the shaking method with less activation.

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

Another alternate that has worked very well is try treating the microglia in the flask with 15 mM lidocaine and use a modification of the shaking method (I have had good success with 15 mM lidocaine for 5 minutes at 37C followed by shaking for 10 minutes at 100 rpm).  Yield and purity are very good, and the protocol is much shorter than the traditional shaking method.