I need to know what are the main markers for identifying macrophages and DC from RAW 264.7 activated in vitro. We intend to observe expression of some surface markers on different phenotypes (macrophages and DCs) of RAW 264.7 activated by flow cytometry, and thus distinguish them from one another.
I don't know if it is correct to say RAW 264.7 cells express F4/80 at all. I have not examined a fresh batch from ATCC. However, to my surprise and disappointment today I discovered that the RAW264.7 cells do not express F4/80 neither do they express CD40, CD80 or CD86, Stimulation with Pokeweed mitogen did not help, but following stimulation with PWM or infection with a Poxvirus they do upregulate expression of IL-6 and TNF alpha. These strange results hold for both IF microscopy and flow cytometry. I am trying CD11b on them next week.
Cell surface markers commonly used in macrophage research
http://www.nature.com/nri/journal/v11/n11/fig_tab/nri3073_T1.html
Dendritic Cell Subsets & Lineage-specific Markers
http://www.rndsystems.com/molecule_group.aspx?g=1334&r=4
RAW 264.7 macrophages and BM Macrophages, express high CD11b and F4/80, compared to macrophages from other cell populations. CD11c, CD40, CD68. MAC1 MAC3 are surface markers that can be expressed at different percentage levels in different cell population. But RAW macrophages specifically express higher CD14 than BM macrophage and other cell populations.
Generally I use CD11c and F4/80 to ID my dendritic cells and CD11b, Cd14, F4/80 to ID macrophages. You can also use Ly6G and Ly6C which are expressed in different levels depending on the role of the macrophage . If you are interested in macrophage phenotypes I would advise also doing LY6C as it is expressed differentially in different macrophage subsets, cells which are high in LY6C are more like inflammatory macrophages that secrete large amounts of cytokines etc, where as LY6C low cells are more patrolling cells.
I am not sure if you can see all the markers prominently stained in RAW cells compared to primary macrophages. Especially if the RAW cells have been passage for too many times. It will be wise to have a base control.
Hi Keyle, I freind of mine is having issues to visualize F4/80 expression when she FACS Raw cells. Do you think that passaging could be an issue?
Thanks
I don't know if it is correct to say RAW 264.7 cells express F4/80 at all. I have not examined a fresh batch from ATCC. However, to my surprise and disappointment today I discovered that the RAW264.7 cells do not express F4/80 neither do they express CD40, CD80 or CD86, Stimulation with Pokeweed mitogen did not help, but following stimulation with PWM or infection with a Poxvirus they do upregulate expression of IL-6 and TNF alpha. These strange results hold for both IF microscopy and flow cytometry. I am trying CD11b on them next week.
Hi Nicholás, can you recall the summary of your research into this topic?
I am encountering the same issues. Lots of good macrophage markers named but none which can be used as an 'activation' marker or indicator of specific polarization with RAW cells. I haven't tried CD14 yet but I fear RAW cells will just express it highly and it will not be a good proxy of activation.
Do you have any advice on how best to phenotype them?
At the moment I am leaning towards ELISAs for IFNg, Griess Assay for iNOS activity, and uptake of fluorescent beads for phagocytosis and MHC II expression (FACS).
Hello, Liam
Unfortunately, I haven't been able to resolve this question yet.
And for now, I'm not working on this. Currently, the project is on stand-by mode.
Best regards
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