I need to do immunofluorescence on tumor section to track different subtype of blood cells accumulated in tumor. Which markers do you recommend? I was thinking about CD11b (for all myeloid cells lines), F4/80 (macrophage), ly6c (monocytes), ly6g (neutrophil), CD19 (B cell), CD3 (T cell). What do you think?
Generally speaking, You may add CD34 and CD38 for stem and progenitor hematopoietic cells from the clinical point of view, but as a laboratory test I'm not sure.
Hey, are you sure about CD11b? My CD charts (from BD, AbD, Janeway) also indicate that lymphoid cells, like NK cells, express that marker. However, I have never dealt with CD11b before. Why don't you look for CD4 and CD8 T cells indeed of just CD3? Just one sample more. ;-) What about MHCII positive cells?
In flow cytometry tumor autofluorescence and non-specific antibody binding can give you significant false positives even with an Fc blocking step. I would recommend a pan-CD45 antibody to gate some of this out. Also, if you're homogenizing a solid tumor you will likely want to add a collagenase + DNase step to release all your cells.
There is no single good myeloid marker, as CD11b can be also be found on NK, T, and B cells subsets and F4/80 won't capture all of your macrophages. I would recommend an MHCII (I-Ad in BALB or I-Ab in B6) in combination with CSFR1 (macrophages/monocytes), CD11c high (DCs), and CD19 (B cells). Ly6c is primarily on inflammatory monocytes, and Ly6g is a good neutrophil marker; however, depending on your opinion of the literature these are also the classic markers for subsetting MDSCs. If your panel is limited, Gr1 will stain both Ly6c and Ly6g, and shares the same epitope as Ly6g (but not Ly6c). I would highly recommend the following article for a thorough how to on how to subset the various myeloid compartments: http://www.ncbi.nlm.nih.gov/pubmed/25446897.
T cells are a very popular in immunology right now, so at a very superficial level CD3 will work (also found on NKT cells), but you will really want to differentiate between CD4 and CD8a T cells. CD4 and CD8a alone are also found on myeloid subsets, but in combination with CD3 should limit you to NKT and T cells. If you really want to get rid of rare NKT cells, include a CD90.2 (wildtype isoform) antibody. NK cells can be important, and NK1.1 is a good marker in B6 mice, while CD335 works in BALBs.
Probably more info/work than you want, but if possible I would recommend two panels for this experiment (lymphoid and myeloid phenotyping). If you let me know how many fluorophore channels you have I can recommend specific panels.
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