Hello everyone,
I am looking at markers that I can use for qPCR to check the phenotype of microglia after primary microglia culture. I found a protocol to prepare microglia from pups but I would like to check their phenotype after culture to be sure to have microglia before starting assays on these cells.
Can you help me ?
Audrey
Hey Audrey,
in our lab we stain primary microglia with Iba1 from WAKO. Alternatively you can also do FACS and stain with CD11b und CD45 (microglia are nir ally CD11b high and CD45 low).
Some people also stain microglia with TMEM119 but this was not working well in our stainings.
Good luck with your experiments,
Anne
Dear Audrey. It is a great idea to check 'who' they are before you start. With PCR you will be able to do relative gene expression which is difficult to relate to the relative proportion of cells, and it is often more interesting to know of X cells - now many are microglia (MG). The new gold standard for MG markers in adult tissue is TMEM119 (or in all tissues Fcrls, not no commercial antibodies for this one). I would suggest that you plate your cells and think about IHC the possibility of using TMEM119 (keeping in mind this is an ADULT microglia marker and I am not sure if you cells are mature enough) or IBA1 (no reviewer will kill you for this, it is a great antibody!) Also do GFAP - as it is these massive suckers that will really contaminate your experimentsn. If you have access to a FACS then this would also be a great way to characterise your cells though - but you will need someone with a bit more experience - and the do permeablised cell staining for markers for MG, fibroblasts and astrocytes. CD11B is useful, and I can't imagine a neutrophil or monocyte making it through the primary analysis, but CD11B will also show them. :-)
Hello Bobbi,
I do have a question regarding the Fcrls as marker for microglia, I have had the impression they are markers for different types of B-cells. Are there also microglia specific Fcrls?
Hi Carsten,
Fc receptor-like S.
Oleg Butovsky has shown that Fcrls a good MG marker also. We have done some validation of our own and found it to be valid. :-)
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