I have treated mice with Ab Ly6-G (clone 1A8), which is known to deplete neutrophils. In the PBL, I can distinguish 2 granulocyte populations: CD11bSSCAhigh, and CD11bSSCA medium (see attached file for illustration). Both of these populations are also Ly6G+. Only one of these populations - CD11b+SSCAmedium - is depleted. Does anyone know what the CD11b+SSCA high population is? Are these also neutrophils? It seems that there are too many for them to be only basophils/eosinophils. Is there a functional difference between the CD11b+SSCAhigh and CD11b+SSClow population?
While I cannot not provide a direct answer to what the populations could be leftover, it could be that your depletion regime (administration route, timing, conditions such as infection, trangenic mice) may not get all the neutrophils.
However, I also believe that a different gating strategy for checking depletion may help tremendously. I would include Ly6c and Gr-1 (Ly6c/Ly6g) antibodies to check depletion. Relying on CD11b and SSC isn't very clear cut imo for identifying granulocytic/monocytic cell types. While I also like to include F4/80 and CD11c, with Ly6c and Gr-1 you can at least crudely separate APCs (Ly6c int-hi Gr-1 neg-int) from neutrophils (Ly6c hi Gr-1 hi).
The CD11b+SSCA hi population is still likely a combination of other granulocytes and monocytes with a higher frequency (following depletion) due to removal of other populations.
Hi, thank you for your answer, Cory!
I agree including other markers could be helpful. However, one problem is that the antibody I use for depletion is directed against Ly6-G. So I am not sure if cells would still appear to be Gr-1 positive, or if so, they may become GR-1 low (because of masking of the Ly6-G antigen; even if cells are not depleted, they may still have bound the Ab).
The depleted population are neutrophils, because they express high levels of Ly6-G; it also fits what I have seen in other papers. On the other hand, I don't think that the SSC-A high population contains monocytes: especially mouse monocytes are not very high in the SSC. Do you have experience with DCs, are they typically SSC-A high?
Thanks so much for your input!
Hannah
PS: At least in my hands, F4/80 does not work very well for flow. Only a very small fraction of CD11b+ cells are F4/80+, and I heard that it is more of an activation marker, rather than a bona fide macrophage/monocyte marker, even though it is widely used in IHC.
I have used 1A8 very effectively to deplete neutrophils and routinely check using a panel of Ly6G RB6-8C5 (which is wiped out completely, and given that I dont use a saturating dose of 1A8 it shouldnt have just bound all antigens), Ly6C (unaffected by 1A8 depletion) and lymphocyte markers (also unaffected).
I also suspect your population may be basophils/eosinophils and the reason you're getting such high proportions is because a large proportion of your myeloid cells has disappeared.
You may have more luck setting the counting gate over the lymphocyte fraction, as these remain constant after neutrophil depletion.
Incidentally F4/80 does work, there's just not many cells in blood as monocytes are F4/80 neg. Works great in tissue.
Dear Peter,
thank you for your input! It's a good point that percentages may be changed due to depletion of the neutrophils.
Best regards,
hannah
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