Kyal Young,

I hope the publication about neutrophil subsets attached is helpful.

Egesa

Article Characterization of Neutrophil Subsets in Healthy Human Pregnancies

Hi ,

You can take a look to this publication:

In vivo inhibition of c-MYC in myeloid cells impairs tumor-associated macrophage maturation and pro-tumoral activities. Pello OM et al. Plos One 2012.

Our collaborators did an amazing flow cytometry analysis of different precursors and immune cells in bone marrow, blood and tumors.

Best luck

Oscar

Thanks Oscar that paper was great! I was struggling the most with panels for precursor cells because there just isn't a whole lot out there on the topic, particularly precursors in the blood!

About your granulocyte panel: Siglec-F is actually a mouse molecule, not human, and it is expressed on mouse eosinophils. To distinguish neutrophils from eosinophils, CD16 is sufficient. Just gate on granulocytes in the FSC/SSC plot. Neutrophils are CD16 positive, eosinophils CD16 negative. Human basophils are in the lymphocyte gate, CD123 positive, class II negative (in contrast to pDC which are CD123 positive, class II positive).

Good luck

Just some thoughts at first glance:

Myeloid/Lymphoid progenitors- no sense in CD20. CD20 is only on mature B-cells, not progenitors. CD19 is on b-cell precursors/progenitors/hematogones.

Granulocytes (Neutrophils, Basophils, Eosionphils) - Agree with Christina that you can distinguish neutros from eos with only CD16 and CD45. But my preferred gating is to gate on total grans in the CD45/SSC gate, then on that gate to place CD45 on y-axis, CD16 on x-axis. If you have a strong left shift, you will see it on the CD16... as a left shift in expression of CD16. Eos are slightly higher in CD45 expression than neutrophils and are CD16 negative. They show up as a nicely distinguished group even with a strong left shift due to the higher CD45 expression. Christina's point about basos is spot on.

Monocytes - CD206? I have no idea about that one. CD11c is on some lymphos, CD11b expressed on neutros and very similar levels to monos. CD64 would be worthwhile.

B cell progenitors-- See note above about CD20. You will not be able to gate on B-cell progenitors without CD19. Perhaps I am misunderstanding your notation and there will be a CD19 in the tube. Then CD20 is useful for gating on maturation stage. CD19/CD10/CD38/CD20 is a really nice combination for B-cell maturation.

Plasma cells -- highly recommended to gate on double positive CD138/CD38. Not clear to me how that last tube will differentiate between the cells you have listed, but I don't know B-regs well at all.

Best of luck!

Thank you both Christina and Elizabeth! I was having quite a bit of trouble in particular with Granulocytes and you guys helped me refine my panels well.

I have taken the rest of the advice on board too, so of my markers are confusing i admit but i am only an honors student and i only get to work with what i have at at hand. but knowing what markers are critical and what wont work at all helps me immensely.

Thanks again!

See Autissier et all, cytometry a, 2010.

http://www.ncbi.nlm.nih.gov/pubmed/20099249