I would guess Neutrophils?!

I think these are early progeny of myeloid derived suppressor cells or MDSC, genarally they are CD11b+, Ly6G+/hi and or Lu6C dim/ bright. Since your population is Ly6G bright it is most likely neutrophil progenitor MDSC. You can stain the population with CD11b and Gr.1  antibody, if you find the same population in similar position and if percentage is comparable then you can be sure that these are MDSCs. I work with MDSC infiltration from BM to inflammation site so I am quite familer with these phenotypic population .

I aggree to sankar. These look like myeloid suppressor cells. Did you block the Fc receptors? because without careful block these can also be monocytes due to the high SSC...

They look like neutrophils to me 

Thanks Sankar and Oliver.  MDSCs had not even crossed my mind.  Sankar, have you ever observed that BALB mice have more of these cells than B6 mice do?

Oliver, I did block, and the entire "live" population ends up with about 1-2% of the single cells being Ly6C hi monocytes, but they fall in with the PMNs. 

Rebecca Jo and Simon, I find a large population of neutrophils down in the much bigger area above the lymphocytes.  It had occurred to me that the 6% might be activated neutrophils though (?).

Thanks all!

I concur with Sankar.  MDSC's tend to look like neutrophils in FACS and are notoriously hard to identify.  They are CD11B+ and GR-1+ (Ly6C high and Ly6G high).  They don't have mature macrophage markers, which is why there are so easy to get mixed up with neutrophils.

In addition, I would suggest that you doing another assay to identify their activity.  My colleague, Dr. Mirzoeva, just presented on this topic yesterday, serendipitously.  She searched the literature and found that people generally use assays that detect their inhibitory qualities (they inhibit T cells, and sometime B cells).  Like if you mix them with a population of T cells, they will inhibit the Cytotoxic T Lymphocyte activity, they'll inhibit production of ROS and arginase, etc.

Also, it's handy to know that MDSC's secrete IL-10 and TGF-B.  So, you could always do an ELISA or qPCR on this cell population and see if they are cranking out these factors.

If you'd like to research MDSC's more, the researcher who discovered them was Gabrilovich back in 2007.  So, if you search his name in Pubmed, you should get a decent idea of what you're dealing with.  Or just search "Myeloid Derived Suppressor Cells".

Anyway, I'm definitely not an expert, but I just wanted to share with you what our lab learned about yesterday and also to say that Sankar has a good point.  Good luck in your research, Michelle!

Michelle , I have not studied MDSC in B6 but balbcseems to have a very sizable amount  of it in BM, if you check spleen you will find the similar MDSC population but percentage would be very low 1-2%. Counter stain your population with F4/80 antibody-if these are MDSCs then they should not show high f4/80 expression. I completely agree with Brian that IL 10 assay would clearly indicate whether these are true MDSCor not but suppression assay could be tricky because generally MDSC only supress antigen dependent T cell activity but tumor associated MDSCs can suppress both antigen dependent and independent T cell activity ( Gabrilovich group, J exp med,2011).

Thank you Bryan, I'll definitely look up some of this information.  They are such "new" cells, that it's hard to consider what their role might be in my cultures.

Sankar, have you tried CD64 staining?  These cells did not show any CD64 at all.

Thanks all!

MDScs arnt CD64+ mostly because in the set up i study them they are neither activated by IFNg nor they are mature macrophages

Thanks Sankar, I should have added that I rarely use F480 anymore and have been using CD64 instead in my assays just as a macrophage marker. So the closest I'd be able to come to the staining you suggest is to use CD64. Thanks! You've given me some excellent information.

Dear Michelle,

in addition to more FACS markers, you could sort the cells and look at them under the microscope. Neutrophils should show the classical lobular nuclear staining and in general their shape and nuclear/granule appearance could give you more of an idea of their identity.

They are really super-high on FSC/SSC, more than I'd expect for neutrophils or myeloid cells. Could they be non-hematopoietic cells, like some kind of stromal cells? Maybe they somehow escaped your gradient? Also for this, microscopic examination could help.

Good luck,

Jakob