Here is what you will find.

CD11b STAINING

Let's assume we are talking about murine blood. In the bone marrow the story changes a little due to the presence of developing cell types. CD11b labels myeloid lineage cells, and will label monocytes, neutrophils and eosinophils. Natural killer cells are CD11b(low), and so if you are using a broad positive gate they will be included.

TISSUE SPECIFIC STAINING

For tissue specific staining, the resident red-pulp macrophages in the spleen are F4/80+CD11b(lo), but resident macrophages the peritoneal cavity they are F4/80(hi)CD11b(hi). Conventional dendritic cells (cDC) are CD11c(hi) and

Seeing as how you work on MS (so I assume murine EAE), is there a possibility you are looking in the CNS? If so, then we can talk in more specific detail.

Gr-1 STAINING

Gr-1 is comprised of two components: Ly6C and Ly6G. Ly6G is exclusively expressed on neutrophils. Ly6C is expressed on a variety of cells, but is most commonly used to distinguish monocyte subtypes. The inflammatory monocyte subset are Ly6C(hi), neutrophils and eosinophils are Ly6C(int), and the 'patrolling' monocyte subset is Ly6C(lo). Therefore CD11b+Gr-1+ cells will include monocytes, neutrophils, and eosinophils. CD11b+Gr-1- will include Ly6C(lo) monocytes. If you are looking in the spleen, then red pulp macrophages and cDCs will appear in the Gr-1(low) area.

Those are the cell types included in your populations. If we are talking about blood/spleen the neutrophils, followed by monocytes, will be the most numerous populations.

EXISTING DATA

If you are trying to look at data that's already been acquired, you can split up your CD11b+Gr-1+ population using side scatter. cDC (spleen, lymph node) will look the same as monocytes so that will be difficult, but they are in small numbers so don't worry too much. CD11b+Ly6C(int)Ly6G+ (Gr-1+) neutrophils are SSC(int), and CD11b+Ly6C(int) eosinophils are SSC(hi). If you are looking at spleen, then the Gr-1- group might have collected some CD11b(low) red pulp macrophages, but a more stringent CD11b gate will exclude them.

NEW EXPERIMENT

If you are planning a new experiment, then as others have suggested, use Ly6C and Ly6G instead of Gr-1, as more information is provided from these markers. CD11c and MHCII will help to exclude cDCs, and Siglec-F is an exclusive marker for eosinophils. However, you can usually gate eosinophils as Ly6G-SSC(hi), where neutrophils are Ly6G+SSC(int), and monocytes Ly6G-SSC(lo); all CD11b+.

This obviously depends on what sort of cytometer you have. If you are using a FACS Calibur, then you have three fluorescence detectors and can run an experiment with CD11b, Ly6C, and Ly6G. If you have a machine with more detectors, then sweet.

Any questions, especially if this is regarding the CNS, then let me know.

- Tom

possibly also dendritic cells depending on the organ investigated.

Some people might also claim that you will get this ellusive myeloid suppressor cells...

CD11b+GR1+ is a typical marker for MDSCs or myeloid-derived suppressor cells. At what context are you looking for the cells? You will find them usually as a subset for tumor-infiltrating lymphocytes. They are majorly immunesuppressive, and are currently attracting a lot of attention as a major cause for tumor-induced immune suppression.

cd11b+gr1+ will be mdsc (neutrophils and other granulocytes) cd11b+gr1- you will have macrophages, monocytes and some DCs

CD11b+GR-1- cells are also considered to be osteoclast precursors

CD11b+ GR1+ cells must be Myeloid-derived suppressor cells which display efficient T cell inhibitory functions and these MDSCs enhance the differentiation of naive CD4(+) T cell precursors into Th17 cells in a highly efficient manner under Th17-polarizing conditions.

CD11b+GR1- cells are mostly macrophages and monocytes.

You would be much better off using Ly6C and Ly6G in conjunction with your other markers. This would be much more definitive. You may also want to look at Tamoutounour S. et. al. Immunity 2013, to more fully define your population.

I would exclude Gr-1 and include antibodies against Ly6C and Ly6G, which will provide you with more information. CD11b+Ly6CHI cells are inflammatory monocytes. CD11b+Ly6Cintermediate cells are primarily neutrophils, which will be shown by Ly6G expression.

Where are these cells from? Blood, spleen etc?

Here is what you will find.

CD11b STAINING

Let's assume we are talking about murine blood. In the bone marrow the story changes a little due to the presence of developing cell types. CD11b labels myeloid lineage cells, and will label monocytes, neutrophils and eosinophils. Natural killer cells are CD11b(low), and so if you are using a broad positive gate they will be included.

TISSUE SPECIFIC STAINING

For tissue specific staining, the resident red-pulp macrophages in the spleen are F4/80+CD11b(lo), but resident macrophages the peritoneal cavity they are F4/80(hi)CD11b(hi). Conventional dendritic cells (cDC) are CD11c(hi) and

Seeing as how you work on MS (so I assume murine EAE), is there a possibility you are looking in the CNS? If so, then we can talk in more specific detail.

Gr-1 STAINING

Gr-1 is comprised of two components: Ly6C and Ly6G. Ly6G is exclusively expressed on neutrophils. Ly6C is expressed on a variety of cells, but is most commonly used to distinguish monocyte subtypes. The inflammatory monocyte subset are Ly6C(hi), neutrophils and eosinophils are Ly6C(int), and the 'patrolling' monocyte subset is Ly6C(lo). Therefore CD11b+Gr-1+ cells will include monocytes, neutrophils, and eosinophils. CD11b+Gr-1- will include Ly6C(lo) monocytes. If you are looking in the spleen, then red pulp macrophages and cDCs will appear in the Gr-1(low) area.

Those are the cell types included in your populations. If we are talking about blood/spleen the neutrophils, followed by monocytes, will be the most numerous populations.

EXISTING DATA

If you are trying to look at data that's already been acquired, you can split up your CD11b+Gr-1+ population using side scatter. cDC (spleen, lymph node) will look the same as monocytes so that will be difficult, but they are in small numbers so don't worry too much. CD11b+Ly6C(int)Ly6G+ (Gr-1+) neutrophils are SSC(int), and CD11b+Ly6C(int) eosinophils are SSC(hi). If you are looking at spleen, then the Gr-1- group might have collected some CD11b(low) red pulp macrophages, but a more stringent CD11b gate will exclude them.

NEW EXPERIMENT

If you are planning a new experiment, then as others have suggested, use Ly6C and Ly6G instead of Gr-1, as more information is provided from these markers. CD11c and MHCII will help to exclude cDCs, and Siglec-F is an exclusive marker for eosinophils. However, you can usually gate eosinophils as Ly6G-SSC(hi), where neutrophils are Ly6G+SSC(int), and monocytes Ly6G-SSC(lo); all CD11b+.

This obviously depends on what sort of cytometer you have. If you are using a FACS Calibur, then you have three fluorescence detectors and can run an experiment with CD11b, Ly6C, and Ly6G. If you have a machine with more detectors, then sweet.

Any questions, especially if this is regarding the CNS, then let me know.

- Tom

further question regarding Ly6G-hi cells:

Thomas, you said Ly6G is a marker specific for neutrophils. Neutrophils should be intermediate in SSC. What if I have additionally to my strong Ly6G-hi SSC-int population Ly6G-hi SSC-hi cells. Are these now activated neutrohpils?

Thomas, in lung what cells are sorted by CD117-GR1+, CD117+GR1- and CD117+GR1+ flow cytometry?

I would like to to know the cell types expressing these. I am studying the different populations of lungs in a murine model of asthma. 

Thanks a lot.

For Cd117 i would suspect mast cells, but I dont know if they also express GR1

Agnes: I think you are correct, Ly6Ghi SSChi are likely to be more activated neutrophils. Some people talk about Ly6G appearing at low levels on eosinophils, but not high levels.

Soledad: Mast cells express CD117 (c-kit) as well as FcER1a, but from memory are supposed to be Gr-1- (i.e. Ly6C- Ly6G-), at least in the steady state. This may well change during certain inflammatory situations, but I'm not familiar enough with those changes to say for sure.

An alternative to mast cells in your CD117+Gr1+ situation could be early forms of monocyte or granulocytes. As stem and MPPs develop into mature myeloid cells, they progressively downregulate CD117 whilst progressively upregulating markers like Ly6C and CD11b (and Ly6G for neutrophils) in the steady state. If you are getting very acute infiltration of myeloid cells, you may be getting early forms of myeloid cells that have retained CD117. In pathology/haematology this is called a "left shift", and happens mostly with granulocytes where metamyelocytes or band cells (the stage before mature neutrophils) can be observed in peripheral blood. It's a bit of a stretch that they would have high enough levels of CD117 to see by the time they are in tissues, but it's certainly a possibility. 

Hello Arza,

I think the best option to isolate these populations would be the Bone marrow. The expression of the antigen CD11b and GR1 (Ly6G) it's very high in both cases. At least it's the experience that I have from my experiments.

Cheers,

Hi Azra,

Just to clarify, are you working with rat or mouse? The antibodies available changes quite dramatically between them (e.g. Ly6G doesn't bind on rat cells, only mouse cells).

In either case, I agree with Francisco, the bone marrow will give you the largest number of neutrophils compared with other tissues (they are in the range of 40% of BM cells). You could also use an intra-pertioneal injection of thioglyocollate -- this is normally used to collect macrophages after 4 days, but after 1-2 days there are stacks of neutrophils. On balance, bone marrow is probably easier.

If you are working in RAT, then a combination of CD11b/c (clone OX-42) and RP-1 will give you your neutrophils. RP-1 is specific for rat neutrophils, and won't bind to other cells or eosinophils.

If you are working in MOUSE then a combination of CD11b (clone M1/70) and Ly6G (Clone 1A8) will give you your neutrophils. The Gr-1 antibody (clone RB6-8C5) will bind to neutrophils at high levels, and monocytes at low levels, whereas Ly6G will bind to neutrophils only. 

For the mouse, immature neutrophils are Ly6G(lo), so if you are concerned with getting 'mature' neutrophils, then stick to Ly6G(hi) cells. There are also some immature cells in the Ly6Ghi gate too which have lower side scatter than the mature ones. You can work this out by plotting Ly6G against SSC. If you compare a bone marrow to a spleen isolation (example attached) you'll see the difference, as the neutrophils in the spleen are mostly mature. You would see a similar affect with RP-1 in rat bone marrow, I would imagine.

RP-1 we got from BD Biosciences (called 'anti-rat granulocytes, clone RP-1), the rest we got from BioLegend.

CD11b will bind to most myeloid cells, and some NK cells (and some peritoneal B cells). In the bone marrow, it will be monocytes, neutrophils, and eosinophils for the most part. Doesn't matter if they are 'activated' or not, they will all have CD11b.

CD48 will come up on monocytes and lymphocytes, it's typically low (or absent) on neutrophils.

Hi, thanks for this question and also for the clear and detailed answers, Tom. I have existing data from mouse mesenteric lymph nodes. Mice were infected with C. rodentium and should be at the peak of inlammation. We have staining with LY6CG, MHC II, CD11b and F4/80. Could you maybe give me some feedback on this gating strategy:

1. I gate on all lymphocytes and then choose LY6CG and CD11b and gate on the double positive population (1.5% only)

2. Within the GR-1 and CD11b positive cells, I look for LY6CG and MHC II and gate on the double positive ( approx 90%).

3. If I then choose GR-1 and SSC,  I am able to distinguish between eosinophils (SSChi), neutrohils (SSCint) and monocytes/macrophages (SSClo), is that right?

4.Further I look within the Macrophage (SSClow) gate for F4/80 positive cells (approx 2%).

Thank you very much for your advice!

Hi Thomas Myles Ashhurst , your responses have been so helpful in the proper interpretation of my previously acquired data using CD11b, GR1, Ly6G, Ly6C, CD11C and F4/80 cell surface markers on bone marrow and spleen cells . Thanks a lot!