I'm working on primary mouse tissues and want to look at the proportion of infiltrating eosinophils in tumours using flow cytometry. Apart from CD45+, MHCII-, Gr1-, CD11b+, CD11c-, does anyone have any more markers to further delineate this population?
Hello
i agree whit researchers , and if you want detect about predominant inflammatory leukocytes infiltrating oral squamous carcinoma (OSC) you can eosinophils incubating in the presence of anti-CRTH-2-PE (MB16, RatIgG2a, Miltenyi Biotec, Auburn, CA)
Good luck
Thanks for your feedback, I've ordered an anti-mouse Siglec-F antibody now to put into my panel above, and will gate for SSChi as well.
Hi,
in general everything is said:
Eosinophils: Siglec-F; Neutros: Ly6G/CD11b/Gr-1; Baso: FcERI
But I would be careful just using Siglec-F against SSC... it depends on your aims. Like mAM also express Siglec-F:
http://www.jimmunol.org/content/183/3/1983.full
beside the paper mentioned above (Hamid), also very useful:
http://www.nature.com/cmi/journal/v7/n5/pdf/cmi201031a.pdf
http://onlinelibrary.wiley.com/doi/10.1002/cyto.a.22012/pdf
cheers
I am not looking at lung samples but mammary gland. The profiles I have been using for macrophages are CD45+CD11b+F4/80+ (and negative for the other markers) and for eosinophils CD45+CD11c-CD11b+MHCII-Gr1-, so if I add Siglec-F I should be able to delineate them okay.
Hi,
Looking at eosinophils in mouse tissues and distinguishing them from macrophage can be difficult. Both eosinophils and macrophage express F4/80 and CD11b (some macrophage).
Even when I compare eosinophils versus macrophage with Gr1 (Ly6G+Ly6C), F4/80, CCR3, Siglec-F, Cd11b, for example and gate with SSC I can find some overlap of macrophage in the eosinophil gating region. The reason I know this for certain is we have several eosinophil-defecient models to use as controls in staining.
I usually gate live/dead, then SSC (macrophage tend to be larger and more scattered in the SSC and FSC parameter), then pusle width to get ride of doublets, then Gr1+ (the range from very low to medium/high), Siglec-F + (some macs can be Siglec-F+ BTW), CCR3+ (but CCR3 expression can be downregulated in tissue for eosinophils in some situations), CD11b+, F4/80med, CD11c-. When I find the presence of this population in WT mice and not in eosinophil-deficient mice, I then backgate to confirm the size of the final gated population is right where I expect eosinophils to be located.
I suggest looking in the supplement Tables of the following: It has a list of eosinophil markers next to macs and DCS...
J Allergy Clin Immunol. 2012 Sep;130(3):572-84. doi: 10.1016/j.jaci.2012.07.025.
I did my best on the tables, but you will want to double check on some of the markers. It is an every changing canvas it seems....
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