I'm running an experiment in whole PBMCs, from which I need to identify CD4 and CD8 T cells, as well as monocytes by flow cytometry. I am familiar with the markers and gating strategies required for T cell phenotyping, but not with those for monocytes. Any resources, ideas?
CD14 is the obvious monocyte marker most commonly used for gating monocytes. But it can sometimes be shed and there are other markers, including a combined CD45 CD64 antibody formulation that claims to gate pure monocytes (Monochromatic gating method by flow cytometry for high purity monocyte analysis. Wong L, Davis BH.Cytometry B Clin Cytom. 2013 Mar;84(2):119-24). Do you need to identify any monocyte subtypes (such as M1 or M2 phenotypes)?
Thanks, Robert. I just need to distinguish them in a preparation of PBMCs.
Appart of the use of CD14, is there any particular gating strategy that should be followed, like first identifying monocytes in a SSC vsFSC plot?
Thanks again
No, don't try sequential gating using intrinsic parameters first (SSC/FSC) followed by fluorescence second (e.g CD14). It is best to use a "heterogeneous gate", where you combine fluorescence and intrinsic properties at the same time. A good example is shown in Fig 1 of the attached publication. In fact, heterogeneous gating is also highly effective for gating pure T cell sub-populations (e.g. combining SSC and CD4).
I use sequential gating to identify B and T cells. Monocytes can be identified as a distinct population in your SSC Vs FSC plot. To simplify your gating in subsequent plots, you can isolate the population of live cells that are more granular and larger than your lymphocyte population. and then look at antibody staining. This is a strategy I've seen some people use. I hope this helps.
There´s a pretty cool OMIP that includes activated monocytes.
Hope this helps
http://onlinelibrary.wiley.com/doi/10.1002/cyto.a.22505/abstract
I would recommend CD45 and C14 for the primary gate. All monocytes are clearly disciminated by this approach
I find gating on CD14+/FSC very useful as it makes possible to identify CD14low monocytes which represent those called "inflammatory monocytes" that also bear cd16 molecule. Interesting paper given by Dr, Landis, thank you.
Hi Enrique,
Have you tried, FSC/SSC gating to identify monocytes in whole blood and then use CD14 and CD16 to delineate monocyte phenotype subsets? CD56 can be used to exclude NK cells.
Cheers
Ahmed
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