You need to post the scatter graph if you want help in gating them.

Also. for the markers, F4/80 is macrophages for mice. CD45 is just basically all leukocytes. CD11b is expressed on both monocytes, granulocytes, some lymphocytes..etc.

Usually, we use the FSC/SSC to gate separate the lymphocytes from the monocytes, and granulocytes. Then use the markers for confirmation and subpopulation analysis.

So... with what markers you have... the most you can do is macrophages, and you might be able to use CD11b for monocytes, but again, this is expressed on quite a few lineages.

Dear Felix Kretzschmar

concerning your question:

In brief, for the gating strategy, please see attached:

https://www.pnas.org/content/pnas/suppl/2015/08/14/1506254112.DCSupplemental/pnas.1506254112.sapp.pdf

Macrophages are often defined as CD11b+/F4/80high, whereas monocytes are defined as CD11b+/F4/80dim, which is a bit arbitrary, without additional markers.

The supplementary figure is from the following paper in PNAS:

https://www.pnas.org/content/112/35/11001.long

Just three general comments to improve macrophage/ monocyte flow cytometric analysis, as I don't know the details of your experiment.

1. Did you stain for live/ dead cells? This will certainly reduce your background, as antibodies randomly stick to dead cells.

2. Did you block Fc receptors on your cells by possibly using purified Rat Anti-Mouse CD16/CD32 (clone  2.4G2) to reduce your background? Macrophages/ monocytes express different Fc receptors on their cell surface and these will bind your antibodies used for FACS with different affinity, if you do not use an additional blocking step before prior to you extracellular staining.

3. Did you use FMO controls to define your gating strategy as laid out in the paper, which is the only way to exactly determine positive staining of your antibodies.

Maybe these considerations help.

All the best & good luck with your experiment,

Michael

Dear Shen-An Hwang

thanks for you quick response. As you wished, I added the two scatters. To which group of cells can i assign the clouds?

Dear Michael Förster

also thanks for the quick response and the attached documents.

Regarding to your comments ... yes, I stained live cells, blocked with CD16/32 and used FMO controls.

Primarily i investigated a receptor on microglia and then optionally i tryed to investigate the receptor on macrophages. However there were different cell types in the scatter.

For my experiment it´s important to differentiate certainly the macrophages, if it´s possible also the monocytes.

Addionally to express assumptions about granulocytes, lymphocytes ...

I know that the antibodies i used, cannot differenciate all different cell types for 100%.

My focus are macrophages and monocytes.

For the bm monocytes -- I would say that the 17.86% is the bone marrow monocytes (CD11b+). The lower cluster might be lymphoid, and should still have CD45. If you use the markers, you should see little to almost no CD11b in the lower cloud.

For the peritoneal macrophages -- My guess would be the higher cluster is most likely macrophages, and if that is true, your F4/80 should be high. The lower cluster might be monocytes, and if that is true, your F4/80 should be lower if not negative. But both should be CD11b+.

Those would be my assumptions.

Dear Felix Kretzschmar

Many thanks for the clarification.

Here are some additional suggestions:

1. Concerning microglia, you can use your markers and define them as live CD45low CD11b+ F4/80+ as per

https://link.springer.com/protocol/10.1007%2F978-1-4939-7837-3_7

In resting/ non-activated state these cells should be Ly6C-, but depending on, which disease you are modelling (in your adult mice) you may find activated CD45int (intermediately expressing) CD11b+ F4/80+Ly6C+ activated microglia.

2. In general, for granulocyte analysis I would add Ly6C and Ly6G to your analysis and define:

Macrophages as live CD45+ CD11b+ F4/80high Ly6Chigh

Monocytes as live CD45+ CD11b+ F4/80dim Ly6Chigh

Neutrophils as live CD45+ CD11b+ F4/80- Ly6Ghigh

Please note that this is a simplified gating strategy, which is not exhaustive for a) all stages of murine development and/or b) all tissues or peripheral/ lymphoid organs and c) different diseases.

All the best & kind regards,

Michael