I am interested in monocyte-derived macrophages from human peripheral blood. From literature, I know I must enrich for monocytes before differentiating them to either M1 or M2. Most of the protocols I have come across use CD14 only beads for the enrichment step. This will largely eliminate the non-classical monocytes (NCM) subset. Are people not interested in NCM subsets or will addition of CD16 add contaminants from other cells? Or are there other reasons? Thanks.
I do not think the reason is that people are not interested in NCM. Most monocyte isolation kits use CD14 beads and hence what you are isolating are the CD14+ classical monocytes. Some companies do sell CD16 microbeads. The reason why CD16 is not used could be that it is expressed in a variety of cell subsets, not just monocytes. Check the CD marker booklet from BD (Page 4). If you want to study all monocyte subsets (CM, NCM and intermediate), you may need to do several sequential +/-selections to get rid of all unwanted populations (as described in the link below).
https://www.bdbiosciences.com/documents/cd_marker_handbook.pdf
http://www.bloodjournal.org/content/bloodjournal/suppl/2015/10/09/blood-2015-06-651331.DC1/Document1.pdf?sso-checked=true
To add to Julie's answer, there are not that many CD16+ nonclassical monocytes. It depends on your experiment and what you want to test.
I agree with Julie Joseph , CD16 is expressed on the surface of Neutrophils too.
NCM are not necessary to differentiate macrophages. These monocytes usually don't leave circulation anyway, so you wouldn't expect your MDM to come from non-classical monocytes.
If you are really interested in them however, I recommend sorting your cells first.
Thanks to all of you for the answers. @Julie, the second link you sent has been very helpful.
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