I usually get two populations of cells, with two peaks in histograms whenever I stain mice splenocytes with this antibody. Can anybody share his experience with me if using this antibody?
Dear Tabasum, I have small experience in staining of splenic macrophages, but we did not see such picture in restricted set of experiments. You can see profiles here, on Figure 2. https://www.researchgate.net/publication/216544249_Accumulation_of_neutrophils_in_the_spleen_of_mice_immunized_with_cells_of_allogenic_tumors?ev=prf_pub
Article Accumulation of Neutrophils in the Spleen of Mice Immunized ...
Tabasum,
Check out this article: http://www.ncbi.nlm.nih.gov/pubmed/22213571
The authors discuss the use and limitation of F480 to identify splenic macrophages.
Eosinophils also express f480, and it is expressed at different levels depending on the type of macrophage.
See this post: https://www.researchgate.net/post/F480
Red pulp macrophages in the spleen are F4/80+ (CD11blo). Eosinophils (Siglec-F+) do express low levels of F4/80, but are easily distinguishable using SSC.
I would recommend gating F4/80 vs SSC. If possible, I would include CD11b, as monocytes express low levels of F4/80.
In agreement with Thomas. Eosinophils will be lower in SSC and can be distinguished that way. However, CD11b is also on eosinophils. Ideal cocktail will contain CD11b/F480/Siglec F if you want to sort the populations using markers. However, the easiest thing will be to use SSC.
Hi Tabasum, The F4/80 ab is marker for mature macrophages , blood monocytes, microglia, Kupffer cells and other macrophages in the connective tissue. The level of expression is different in different type of cells and varies depending to the state of maturation, developmental processes, adhesion and signaling function of cells. The level can be up or down modulated also depending of the tissue localization and cell activity. I do usually FACS staining of splenocytes, blood, Bone morrow or non lymphoid cells (brain, lung) with F4/80 /CD11b/CD11c/Gr1 and I get a nice profile, gating on these markers (exemple Cd11b VS GR1 in dot plot/region on sigle or double positiv cells/ monocytes or neutrophiles)-to see in histogram F4/80 expression. You have to Us the isotypic control because he gives same nonspecific stain. I us F4/80 Pe from Cedarline Clone C1 A3-1 ( rat IgG2b) and Alexa Fluor 647 from Molecular probe (rat IgG2a). Hoping these details can help you. Good luck.
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