Trying to figure out which markers I should detect to distinguish (if applicable) activated monocytes (from PBMCs) and macrophages.
I assume the activated monocytes should still preserve their surface markers such as Ly6C, CCR2, etc. if they lose these markers and gain other markers such as CD11c, MHCII (or F4/80) then they are differentiated to DC (or macs).
Hi all, thanks for replies! I'm working with peripheral blood derived monocytes, but in naive state they didn't work for my experiment. Thus I decided to introduce PMA. Now the trick is in recognizing monocytes/macrophage subsets.
Olga, that very much depends on the species. CD14/CD16 are in general good indicators for monocyte/macrophage differentiation, as well as expression levels of MHC class II and co-stimulatory molecules. It also depends on what you are using to stimulate your cells with. HAve you tried LPS?
Hi Olga--you may want to look at the expression of L-selectin (CD62L) as well as HLA-DR. Circulating monocytes should be L-sel high/+ and have moderate expression of HLA-DR. I believe that after activation, monocytes should lose their L-selectin expression, although I have not definitively tested it after in vitro culturing. You should definitely see higher expression of HLA-DR/co-stim molecules. LPS or IFN-g are considered a good maturation stimulus. Good luck!
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