I have stained mouse BM with F4/80, CD11b, MHCII, CD206 and CCR7. Cells were Fc-blocked. When gated on F4/80_hi/CD11b_med-hi, I see a population that is CCR7+/CD206+ double positive (see attached). These cells are also MHCII+. Why are they double positive? Aren't these two markers should be mutually exclusive on M1 and M2 macrophage populations?
Also I see a population which is CD206+ MHCII+ F4/80+ but CD11b-. What are these?
Hi, there can be several issues to consider. First, are the mice healthy or you are working on a disease model? Because in case of many inflammatory diseases, BM, especially its myeloid compartment changes significantly .
Here are some comments that might be helpful:
1- Even though it is accepted as a pan-mq (or even a pan myeloid cell) marker not all F4/80 macrophages are CD11b+ (especially in case of cancer and certain specific compartments).
2- Other cells such as NK cells are also express CD11b albeit at lower levels than that of myeloid cells. Be sure that your CD11b gating is stringent enough.
3- Markers of M1-type macrophages are also shared by DCs, therefore CCR7 or CD11c can also indicate the presence of DCs.
4- There is no clear distinction between M1 and M2 phenotypes although certain markers have been associated to them. As you are working on a primary lymphoid/hematopoietic organ, and i assume you have repeated the experiments and obtained reproducible results, one can presume that these cells are somewhere between M1/M2 phenotype differentiation/decision.
5- I would suggest to add some other markers like CD11c and CD163 to your panel.
I hope these will help, good luck.
Thanks for the answer. These are NSG mice. They for sure have macrophages but with somewhat impaired functionality and I do not know what should be the marker profile on them. I am not even sure whether I should see any activated macrophages in vivo in healthy unchallenged mice, especially NSG. Currently I am growing BMDMs to activate them in vitro and see what the readout will be. Maybe what I am seeing is actually some CD206lo/CCR7lo population.
I will include CD11c in the next staining. But I thought that numbers of DCs in the BM should be quite small. Regarding CD163 I wanted to include it but couldn't find any anti-mouse conjugate and don't want to deal with unconjugated ones as the final staining will be complicated enough. Shall I try CD86/Arginase instead of CCR7/CD206?
Also I am planning to include IC staining for CD68 as a general macrophage marker.
I have attached more detailed FACS plots for all MHCII/CD11b subpopulations.
Well, i have no experience with the BM of NSG mice but regarding the second plots you sent, my suggestions are below.
The population in the gate A seems to be mostly granulocytes, you may need to exclude Gr1+ (especially Ly6G+) cells. I believe then you can have a more clear view. Even you might try to use a dump channel to be used for Lin-positive cells.
CD68 can be a good idea to support findings since staining with F4/80 is generally problematic due to its low-expression (dim) pattern.
I would first do these, then think about using alternative M1 or M2 markers
Just out of interest, have you tried to do a fluorescence minus 1 control (stained your samples and gated with F4/80 and all markers except CCR7/CD206)? When I see a diagonal pattern like yours on my samples, it sometimes may be the natural autofluorescent background of the cell.
That was a new panel, so I did FMOs for every stain. It does not seem to be a compensation issue. Does not look like spread either, a spread can't be that bad.
I know there is an issue with autofluorescence of activated macrophages, but it is supposed to manifest mostly in blue diapason, so these stains should not be affected.
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