I am trying to verify the effects of an new peptide adjuvant on dendritic cells.
There is a large number of sub-populations of dendritic cells found in the spleen, as in most other organs.
These subpopulations are not all CD11c +, and there is a large number of molecules that can characterize each. But it still does not exist an antibody capable of specifically identifying all.
Maybe the article by N. Rochereau could be very useful: Rochereau, Vaccine 29 (2011) 3655-3661.
Regarding the 33D1 antibody (Nussenzweig 1982), it mainly stains Langerhans cells, whereas CD205 antibody (dec205) stains mostly interdigitating DC in lymphoid organs. I'm not sure that 33D1 binds to CD205.
other markers are most useful:
CD207, CD208, CD209, CD303, CD304
CD301, CD283, CD287, CD288
CD123-Mip3a/b-MMP12
And especially CD317
Hope this would be helpful.
Hey Catarina,
Thank you for your answer. Yes, I wanna look at co-stimulatory molecules , MHC levels as well as the cytokines production. Do you also put other markers for gating? I used B220 and CD11c for gating and I check CD86, MHCII and cytokine/chemokines.
Hi, in addition of what has been said above (CD11c, MHC II and costimulatory molecules…) it could be useful ( and easy) to measure the frequency of CD11c positive MHC II intermediate and MHC high populations after treatment. Your adyuvant should up-regulate costimulatory molecules levels (CD86, CD80, CD40) but is could also increase the maturation level of the semimature (MHC II intermediate) DCs pool in the spleen.
For gating CD11c is almost enough. You will notice that they´re not very frequent and athey also express MHC II.
Hi,
I use also 33d1 (cd205) marker for dendritic cells.
If you want to be absolutely sure you are gating DCs you should probably use more than one marker.
I was recently referred to this review :
http://www.nature.com/nri/journal/v12/n4/fig_tab/nri3175_T1.html
Hope this would be helpful.
There is a large number of sub-populations of dendritic cells found in the spleen, as in most other organs.
These subpopulations are not all CD11c +, and there is a large number of molecules that can characterize each. But it still does not exist an antibody capable of specifically identifying all.
Maybe the article by N. Rochereau could be very useful: Rochereau, Vaccine 29 (2011) 3655-3661.
Regarding the 33D1 antibody (Nussenzweig 1982), it mainly stains Langerhans cells, whereas CD205 antibody (dec205) stains mostly interdigitating DC in lymphoid organs. I'm not sure that 33D1 binds to CD205.
other markers are most useful:
CD207, CD208, CD209, CD303, CD304
CD301, CD283, CD287, CD288
CD123-Mip3a/b-MMP12
And especially CD317
Hope this would be helpful.
Dear Chun-yu Tung,
Spleen DCs are divided in to two major subtype, one of them CD11c+ CD11b+ CD8- and the other one CD11c+ CD11b- CD8+. If you have a chance to use FACS Canto, you can investigate different DCs subtype co stimulator receptors.
Good luck
Hi Chun-yu,
The key to making it "easier" to see by FACS is to use a "dump" channel to gate out unwanted populations (T cells, B cells, etc). You can do this by using for example, Anti-CD3-Biotin, CD19-Biotin, etc. and then stain with your antibodies of interest (CD11c, CD11b, etc) that are directly labeled with color. This allows for best resolution of your population with limited FACS machines while gating out the streptavidin channel. We use this gating strategy routinely at iQ Biosciences for immune monitoring of minor populations such as dendritic cells. The key is to research each marker carefully. For example, B220 is typically thought of as a B cell marker however, it is expressed on a subset of plasmacytoid dendritic cells so this should not be used, etc.
Best Regards,
Omar
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