I usually look at microglia isolated from mice brain using the standard percol gradient method without using Collagenase/DNAse steps i.e. just meshing the brain with 10 ml syringe plunger and then layering 30% brain cell suspension on 70% percol. This is the way I look for infiltrating lymphocytes (CD45 Hi) and microglia (CD45 Int CD11b+ cells) by Flow cytometry. The method works well for analyzing infiltrating lymphocytes but for microglia, I get the fluctuating data for the mice even within the same group. Is there any other clean and better way to isolate and characterize microglia from mice brain? Waiting for your suggestions.
Dear Tabasum
There are specific products for microglia isolation which can save you lots of time and make your life easier.
Here's a tutorial video which might be helpful for you. The entire procedure is shown step by step for three adult mouse brains, and it is the same for human brain tissue. For young mice until P7 you can skip the myelin removal step. For a small number of mice the enrichment may also be done manually with a magnetic separator.
Kind regards
Sandra
https://youtu.be/XYg7KSsZZys
Dear Tabasum
There are specific products for microglia isolation which can save you lots of time and make your life easier.
Here's a tutorial video which might be helpful for you. The entire procedure is shown step by step for three adult mouse brains, and it is the same for human brain tissue. For young mice until P7 you can skip the myelin removal step. For a small number of mice the enrichment may also be done manually with a magnetic separator.
Kind regards
Sandra
https://youtu.be/XYg7KSsZZys
You can use immunochemical method to identify them because there are numerous
antibodies against microglial markers: anti- ferritin, CD68, the major histocompatibility complex class II (MHC II), etc. (see Wojtera et al., Folia Neuropathol 2012; 50 (1): 74-84 and Folia Neuropathol 2005; 43:311-321.
Perform imunohistochemistry and use CD11b anitbody to identify activated microglia.
Thanks everyone for the response. The issue is that if I can get enough of these isolated microglia and sort them by FACS sorting for downstream applications?
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