Hi everyone! I will isolate microglia from adult hippocampus by using Miltenyi protocol with Adult Brain dissociation kit and Cd11b microbeads. I will perform the success of my isolation with CD45 and Cd11b antibodies for FACS and then I will plate the cells on coated coverslips (30' UV and then washed with h20 for 3 times) with polylisine for immunocytochemistry experiments in DMEM-F12+GlutamaX 10% FBS 1% pen/strep, 1% gentamicin and 10ng/ml of MCSF.
I'm thinking to stimulate cells not more than 48 hours and changing at the same time of the stimulation.
What do you think about this protocol? Do you think it will work well?
It's the first time that i try to isolate microglia from a specific brain area :)
This protocol is a time-tested method. But in my experience it should give you both macrophages and microglia. I used FACS using Iba-1 to sort out embryonic microglia.
From my experience, I would recommend plating the cells directly after the CD11b MACS sort. The additional FACS sort will make the culture only marginally purer. Additionally, the cells will be overstressed and likely won't plate well. Also consider the relatively low numbers of cells you may yield from a single hippocampus. A whole mouse brain can reliably yield 100,000-150,000 cells (via percoll centrifugation and MACS sorting), although I am not familiar with yields from the Miltenyi Dissociation kit. I recommend having a look at this recent pub for more information: Article Isolation and Culture of Microglia
Good luck!