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.

Hmm, did you separate out the FMO staining by your gates A to E? The reason I'm interested in this is because gates D and E appear to have the same background level as each other, but look separate from A to C (so the quadrants for positive staining you have set may not be true for D and E). 

Mertk is another validated pan-mouse macrophage surface marker that you could also use to confirm CCR7 and CD206 expression (http://www.nature.com/ni/journal/v13/n11/full/ni.2419.html). I've tried the biotinylated antibody and it works well, and its addition should persuade most people in the field that these are macrophages and not DCs (CD11c as other people have suggested is also a good one to add to confirm that you have ruled out conventional DCs). 

Apparently CCR7 may be seen on some macrophages, so it is possible that you have also found an interesting phenomenon (maybe superimpose the FMOs for each gate on top of the positive staining for each gate during data presentation to best show this)!