DC phenotyping - Markers for DC (Oct/21/2008 )
FCM for phenotyping DC.
What markers do U recommend in what combination?
mouse or human?
there are different subsets in different models. myeloid, plasmacytoid, langerhans, CD8+ DCs, CD4+ DCs, immature, mature ... take your pick.
commonly used markers for Human DCs are CD11c, HLA-DR, CD123, CD80, CD83, CD86.
common markers in mice include CD11c, CD8, CD4, Class II (I^b in B6 mice), CD80, CD86
Thx JUI, I am interested in human DCs
commonly used markers for Human DCs are CD11c, HLA-DR, CD123, CD80, CD83, CD86.
With CD11c-HLA-DR-CD123 combination do U gate lin neg cells or not? And this combination better or BDCA-1,BDCA-2 better for phenotyping myeloid/plasmacytoid DCs?
For human dendritic cells from PBMC, you will find two major subsets - myeloid and plasmacytoid - which function in different aspects of immunology. in order to identify these subsets by flow cytometry, use CD11c, HLA-DR, and CD123. You are correct to use lineage markers for a "dump gate." The lineage markers should include (but are not limited to) - CD3, CD4, CD8, (CD14?), CD19, CD56. I think that I am missing a couple (it's been a few years since I have specifically stained for these populations and the strategy may have changed a bit).
from your standard scatter plot, gate out doublets, then SSC-A vs lin, gate Lin-. mDCs are lin-/DR+/CD123-/CD11c+. pDCs are lin-/DR+/CD123 bright. i think that there is some literature of a small population of human DCs that express CD4 but you should look into this.
the BDCA molecules are primarily used in MACS separation to purify subsets of DCs, and i'm not entirely clear as to why these molecules haven't transitioned to mainstream flow cytometry. maybe someone else can comment.
good luck!
ps. the mDC comprise the bulk of the DC pop in PBMC with pDC as a much smaller percentage. i would recommend using CD123-PC7 from BD, this is a very nice looking stain.
JUI, Thank U. That was so clear.
I am not just new to DC but doing little bonus experiment related to human renal DCs without previous any knowledge and needed some tips to follow. I have gone through a lot of papers just to understand the rigth markers. What U have mentioned is exactly what I could understand till now and U have confirmed it. I am omitting CD56 from the lin markers as I came across some small subset of DCs expressing CD56.
I initially thought using BDCA as I can then design different combinations of other markers for phenotyping more freely as other colors in FCM but as I said it is not a major experiment I plan to do, I am using the other combinations as we already have them with us and just need to purchase CD123. If the results are interesting and convincing then may be think of BDCA also. BDCA is little confusing to me. I will be using fresh DCs so need not worry but came to know that within few hours of culture BDCA2 is down regulated. There is some population that express BDCA3 also and have not understood about them till now and was looking into it. BCDA4 and BDCA2 are expressed in similar cells so I think I need not worry about BDCA4. But, wondering what are mDCs expressing BDCA3 and if they express BDCA1 as well or just BDCA1.
And, yes, have ordered for CD123-PC7 from BD. 
I think I am in right track now.
Thx a lot.
Glad that I could help!
It is interesting that you have found CD56 on DCs. CD56 is an activating NK cell receptor (from the same family as NK1.1 in mice) that I could have sworn was exclusively expressed on human NKs. Not even activated CD8+ T cells, which often upregulate NK cell markers, express CD56.
I would not be totally surprised if this is actually true.
It is interesting that you have found CD56 on DCs. CD56 is an activating NK cell receptor (from the same family as NK1.1 in mice) that I could have sworn was exclusively expressed on human NKs. Not even activated CD8+ T cells, which often upregulate NK cell markers, express CD56.
I would not be totally surprised if this is actually true.
That was not my finding but I read in a paper about these subsets; so in my protocol I excluded CD56 in lin marker. I have not looked into detail about them as my first concern is to find markers and run a pilot till I am determined to explore more.
Could you send me the citation?
I was only able to find one paper that reports CD56 expression on wild-type human DCs. This paper, which came out the beginning of this year, uses monocytes cultured in the presence IFNa and GM to generate the so-called cytotoxic DC. This subset has been found and confirmed in mice. The authors fail to show that this subset is present in human PBMC.
In terms of your Lineage markers, it honestly shouldn't make a difference if you don't add CD56. NK cells do not express the IL-3 receptor (CD123) nor do they upregulate Class II (as far as I know).
I was only able to find one paper that reports CD56 expression on wild-type human DCs. This paper, which came out the beginning of this year, uses monocytes cultured in the presence IFNa and GM to generate the so-called cytotoxic DC. This subset has been found and confirmed in mice. The authors fail to show that this subset is present in human PBMC.
In terms of your Lineage markers, it honestly shouldn't make a difference if you don't add CD56. NK cells do not express the IL-3 receptor (CD123) nor do they upregulate Class II (as far as I know).
May be that was the same paper I came across - I will go find the paper and let U know.
This paper says 1-4% of circulating DCs can be CD56+.
Burt BM, Plitas G, Nguyen HM, Stableford JA, Bamboat ZM, DeMatteo RP. Circulating HLA-DR+ natural killer cells have potent lytic ability and weak antigen-presenting cell function. Human Immunology. 2008 Aug ;69(8):469-474.
And may be this is the paper U were mentioning about.
Papewalis C, Jacobs B, Wuttke M, Ullrich E, Baehring T, Fenk R, et al. IFN-alpha skews monocytes into CD56+-expressing dendritic cells with potent functional activities in vitro and in vivo. J Immunol. 2008 Feb 1;180(3):1462-70.
I have to read them both once before I can comment. But, leaving these here for reference.