It should be better if you wait they attached on the dish colture and stop to be round cells, generally it takes 48h with primary microglial cells

Do you usually coat the wells they are plated in? I've been growing them for about 5 days in culture and they are still small round cells with no microglial morphology. I'm wondering if they are healthy

Hi Antwoine,

I usually wait until primary microglia have grown for at least 3 days in culture before attempting analysis and usually let the cells adhere for 1 day before beginning experiments. In my experience primary neonate microglia are generally adherent within an hour of plating and should start to show typical morphology not long after. Small round cells would suggest unhealthy/dying cells. I'd suggest trying a few different substrates including uncoated glass. 

Hi Antwoine,

I plate them on polylisine coated dishes or glasses, otherwise they do not adhere and grow.

Thanks, I am going to plate them on poly L lysine this time around and see if I get better survival

Hi Antwoine, I work with rat primary microglia isolated from mixed glial cultures. I plate them on uncoated plates or coverslips and they adhere well within the hour. I always wait 24 hours before adding the drugs. In my experience, microglial cells do not divide much and do not survive for many days after plating if they are not stimulated, so I would't wait more than 48 hs to perform the experiments.

Hi Antoine,

I also culture microglia from adult mice using a magnetic bead extraction. As above, poly-l-lysine coated plates/cover slips are very important. For the adult cells, it appears they need around 5 days to be fully process bearing. I add M-CSF enriched media but they also culture well without. I use the cells at 7 days post extraction.

Andy

Hello Antwoine,

in my opinion, basically, it depends on for what purpose do you want to use the cells.

For example I start some stimulation experiments 48 hours after planting microglia into 6-well plates onto poly-L-lysine coated slides (to improve their adherence). The 2-day pause is needed so that cells "calm down" after isolation from neonatal pups. If I started my experiments earlier, the cells would produce some "stress response staff" even being unstimulated - just because of "mechanical stimulation" originating in isolation process.

On the other hand, I do not have good experience with cultivating microglia for longer than 3-4 days. They do not grow well until you add some "suplements" like M-CSF (as mentioned above). I've read that you may also  support them using astrocyte-conditioned medium. But to be honest, I am not into long-term cultivation of microglia alone ;-)

Good luck with your work!

PS: Judi's comment on the dependence of morphology on the amount of cells on slide is nice. Do you, please, have any reference on this topic?

Thank you guys a lot, i've integrated your answers and it seems that I was not plating them in high enough density or using coated plates.  After about 10 days in GM-CSF treated media the cells began to show processes.  But this was brought down to 48 hours when I plated at a higher density "100k per well in 6 well plate" On a poly l lysine coated plate.  It is important that they return to resting state because i'm examining the stress response difference between young and old animals.  thank you all for your answers.

Hey,

I have been working with primary microglia for some time, I can tell you what we are doing.

1. for in vitro experiment (like stimulation..) we only use microglia from shake-off culture method, you may know the protocol for neonatal culture, for the adult, it is more difficult and complicated and you can find it in a recent publication: Panell et al GLIA 2015 

2. for MACS sorting cells, basically we do not use them for further experiments in vitro because:1)they are quite expensive and it is hard to yield a lot of cells 2)the purity of these cells are not as high as shake off cells if you only use 1-2 columns 3)they are hard to grow in vitro, as you mentioned in your question.

Good luck!

Hi Antwoine,

You should probably note that using GM-CSF (classically used to obtain mature dendritic cells from bone marrow) may not give you a transcriptional profile that is close to freshly isolated microglia, as using M-CSF (classically used to obtain mature macrophages from bone marrow) would.

See this paper Butovsky et al 2014 Figure 6

http://www.ncbi.nlm.nih.gov/pubmed/24316888

I find plating at around 400,000 microglia (one adult mouse brain) per well in a 6 well plate gives the best mircoglia at 7 days. 

Hi everybody: How often should I perform media change to microglial cultures prior to experimental treatments? Should I perform full media change 24 hr after isolation to remove cell debri, dead cells?]

I have heard people culturing microglia in GM-CSF for 5 days to have them fully rested (ramified), then GM-CSF removal for 2 days, prior to experimental activation, but would like to confirm that.

I have successfully isolated microglia using magnetic beads from neonatal mice and the cells are being kept in DMEM:F12, 10% FBS, 1% Glutamax, 1% Penn/Strep and 10ng/ml GM-CSF.

Thank you in advance,

Rafael.

Hi Rafael,

when I isolate microglial cells from primary glial colture or from brain tissue using magnetic beads, I usually keep them in culture for 2 days before performing any experiment.

Good work!

Clotilde