I did a bone marrow dendritic cell differentiation (7days) using Gm-csf at both day 0 and day 4,
the question is, Which CD is more specific marker to check ex-vivo Dendritic cell differentiation using flowcytometry?
i am wondering whether CD80, 86, 40, MHCII and CCR7 will be the best option?
or CD11c, CD123?
or there is a more specific marker to validate the differentiation?
Hi Moustafa,
There is a long list of DC markers for their immunophenotyping characterizations.
For immature DC, you can go for the CD80/86(positive) and CD83 (negative) on the 6th day before the antigen exposure to see the expression and number of your iDCs in cell culture to calculate the antigen concentration for the exposure.
and the most important thing, you should check the mannose receptor markers DEC20/CD205 as they are the endocytic markers which will be overexpressed at the immature state of DCs.
For mature DCs, you can use CD83(positive), which will tell about the maturity of the dendritic cells on the 8th day, with CD80/86(positive) as a co-stimulatory marker. CD40(positive) will tell about the activated DCs, MHCII(positive) can tell about the antigen presentation to T cells. CCR79 (CD197-positive) will tell your mature DC is able to migrate or not.
CD209 DC-SIGN also a trusted marker for DC maturation and activation.
you can use CD11c and CD123 as well.
Moreover, it also depends on which method and which type of antigen you are using for the antigen exposure, whole tumor lysate, mRNA, recombinant protein or nanoparticles, and which endocytic receptor you are targeting for the endocytosis.
As you know this is very big area of immunotherapy.
Hope you will get some points.
Thank you.
CD11c. For more detailed characterization, follow Sameer Varma 's suggestions.
Dear Moustafa,
CD209 (DC-SIGN) as well as CD11c would be most appropriate marker for DCs.
Otherwise, there is a long list of CD markers used for DC phenotyping.
All the best.
My main suggestion would be to switch from GM-CSF to Flt3L induced BM-DCs. GM-CSF "DCs" are a mixture of Macrophages and Dendritic Cells (See Helft et al. Immunity, 2015), Flt3L actually makes the bone marrow differentiate into cDC1s, cDC2s and pDCs that express most of the appropriate markers as well as transcription factors, and the cultures are practically devoid of macrophages, which can really skew the results of some assays.
If you want to stick to the GM-CSF, I'd still recommend that you at least look at the paper I cited above - it will give you a good idea what markers to use, and what your FACS plots should look like.
Good luck!
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