Approximately 1-10% of peripheral blood mononuclear cells (PBMCs) in human blood are dendritic cells. This scarcity, along with a relatively low abundance of unique markers, and the continuous discovery of novel subpopulations, has made it a challenge to isolate DCs for functional studies. To address this challenge, R&D Systems has now developed a one-of-a-kind negative selection kit to enrich for DCs isolated from human blood.
Unlike positive selection kits, the MagCellect Human Blood DC Isolation Kit (Catalog # MAGH110) leaves the isolated DC population untouched, reducing the risk that the cells will be altered by the selection process. Flow cytometric analysis of the DC population isolated with the MagCellect Kit demonstrates that the cells are over 90% CD14-CD19-BDCA+BDCA2+BDCA3+ (Figure 1). In addition, functional analysis of the total enriched DC population confirms that the isolated cells maintain their ability to stimulate the proliferation of allogeneic T cells in a mixed leukocyte reaction assay (Figure 2).
|Figure 1. Analysis of Dendritic Cells Isolated Using the MagCellect Human Blood Dendritic Cell Isolation Kit by Flow Cytometry. The MagCellect Human Blood Dendritic Cell Isolation Kit (Catalog # MAGH110) was used to enrich for total dendritic cells (DCs) obtained from human blood samples. Different DC subpopulations were detected, before (left) and after (right) enrichment, by flow cytometry using the indicated antibodies. (A) Total DCs were detected using CD14, CD19, CD1c/BDCA1, DLEC/BDCA2, and Thrombomodulin/BDCA3 antibodies. CD14 and CD19 are markers of monocytes and B cells. (B) Plasmacytoid DCs (pDCs) were detected using DLEC/BDCA2 and CD45 antibodies. (C) CD56+MHCII+ myeloid DCs (mDCs) were detected using CD56 and MHCII antibodies. (D) CD16+CD11c+ mDCs were detected using CD16 and CD11c antibodies. Percentages of the obtained subpopulations may vary due to donor variability.|
|Figure 2. Dendritic Cells Isolated using the MagCellect Human Blood DC Isolation Kit Induce Allogeneic T Cell Proliferation. Dendritic cells were isolated from human blood using the MagCellect Human Blood Dendritic Cell Isolation Kit (Catalog # MAGH110). The ability of these cells to stimulate the proliferation of allogeneic T cells was assessed using the mixed leukocyte reaction assay. T cells were cultured either alone (orange line), or with serial dilutions of isolated DCs (blue line), or cultured monocyte-derived DCs as a positive control (green line), starting at a T cell:DC ratio of 8:1. T cell proliferation was measured after 3 days by 3H-thymidine incorporation.|
New Protocol for the Enrichment of Untouched Human Blood Dendritic Cells
The American Association of Immunologists (AAI), Baltimore, Maryland, May 7-11, 2010