I am having problems in my sortings of CD8+ DC from spleens of chronically infected mice. I did a gene expression array and, after analysis, I identified a lot of T cell markers (CD3e, Thy1, CD8b, TCR) in my sorted CD8+ DCs from infected mice (which I don't see in my naive mice). I used anti-CD3e PerCP-Cy5.5 to select CD3- CD8+ DC, and I used EDTA 10 mM for 5 min followed by an EDTA 4 mM MACS buffer the rest of the time until sorting the cells (and I did a dump channel for doublets as well). But I still see those T cell marker genes. Any suggestions for how to make a better single cell suspension or a good surface staining antibody to discriminate between single DCs from DCs-T cells?
Hi Daniel,
If you stain your cells against DC-SIGN (CD209), which is a dendritic cell marker, you will be able do discriminate between CD209 negative and positive cells. Thus, CD209 negative cells will be lymphocytes and CD209 positive cells will be DCs.
There are other specific markers of DCs you can use, such as CD1a or Mannose Receptor. If you want to be sure, you can stain your cells against two or three of these markers and select the negative cells.
I hope this helps.
Cheers,
Paulo
Hi Paulo, Thanks for you attention, but I am looking to a stable marker that I could identify in T cells that I could dump T cell that interacts with DCs, It seems that interaction between T cell and DCs is so strong that I can't disrupt just using EDTA,
If you want to disrupt the interaction, perhaps you could trypsinize the cells and make a single cell suspension.
But the epitopes for CD11c, MHC-II and CD8a would be intact for staining with antibodies afterwords?
Use always EDTA 10 mM, exclude doubles by FSC-A vs. FSC-H analysis (discriminate doublets from single cells. In that plot, everything that is out of the diagonal is considered as a doublet) and include thy1 in your dump channel. I hope this helps.
After EDTA treatment sort the cells using flow cytometer based on CD4 and CD11c sating instead of magnetic sorting...and exclude double positives. It will require a large number of cells but its worth a try.
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