Dendritic cells (DC) provide critical for innate and adaptive immune responses. These specialized cells have the ability to capture, process, and present antigen to native T cells to promote their differentiation and activation. Although all dendritic cells are capable of presenting antigens, they are a heterogeneous population of cells. Several dendritic cell subsets have been identified that differ in location, phenotype, and immune function. This plasticity allows them to differentially form an immune response when presenting different microbial antigens or damaging cellular components.
About 1-10% of peripheral blood mononuclear cells (PBMC) in human blood are dendritic cells. This scarcity, as well as the relatively low abundance of unique markers and the newly discovered new subpopulations, make the functional study of dendritic cells a challenge. To solve this problem, R&D Systems has now developed a negative selection kit that enriches dendritic cells isolated from human blood. Unlike the positive selection kit, the MagCellect Human Blood DC Isolation Kit (catalog number MAGH110) allows the isolated dendritic cell population to be untouched, reducing the risk of cell changes during sorting. Flow cytometric analysis of dendritic cell populations isolated with the MagCellect Kit confirmed that more than 90% of the cells were CD14–CD19–BDCA1+BDCA2+BDCA3+ (Fig. 1A). 2-8% of them were BDCA2+CD45+ plasmacytoid dendritic cells (pDC; Fig. 1B), and 82-88% were myeloid dendritic cells (mDC). 20-40% of the mDC cell population is BDCA1+CD11c+, 20-40% is BDCA3+CD11c+, 10-20% is CD16+CD11c+, and 10-25% is CD56+MHCII+, indicating that the MagCellect Kit can be isolated and identified. CD56+ and CD16+ mDC subpopulations (Figures 1C and 1D).
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Functional analysis of the enriched population of all DC cells confirmed that the isolated cells maintained the ability to activate the proliferation of allogeneic T cells in a mixed leukocyte response (Figure 2). Since the isolated dendritic cells are not touched by antibodies during the entire enrichment process, they are likely to retain more functional properties than the cells isolated by the positive selection technique. In addition, the MagCellect Kit labels unwanted cells with a mixture of two antibodies, eliminating the need for a separation column and making it easier to use than other kits. R&D Systems' new MagCellect Human Blood Myeloid Dendritic Cell Isolation Kit (catalog number MAGH120) also has similar advantages.
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Figure 1. Analysis of dendritic cells isolated from the MagCellect Human Blood Dendritic Cell Isolation Kit using flow cytometry. The MagCellect Human Blood Dendritic Cell Isolation Kit (catalog number MAGH110) can enrich all dendritic cells (DC) from human blood samples. Different dendritic cell subpopulations were detected by flow cytometry using the indicated antibodies, before enrichment (top panel) and after enrichment (bottom panel). A. Detection of all dendritic cells using anti-CD14, anti-CD19, anti-CD1c/BDCA1, anti-DLEC/BDCA2 and anti-Thrombomodulin/BDCA3 antibodies. B. Plasmacytoid dendritic cells (pDC) were detected using anti-DLEC/BDCA2 and anti-CD45 antibodies. C. Detection of CD56+MHCII+ myeloid dendritic cells (mDCs) using anti-CD56 and anti-MHCII antibodies. D. Detection of CD16+CD11c+ mDC using anti-CD16 and anti-CD11c antibodies. The percentage of subpopulations tested may vary from donor to donor.
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 number MAGH110). The ability of these cells to stimulate proliferation of allogeneic T cells was assessed using a mixed leukocyte reaction assay. T cells were either cultured alone (gold wire), or serially diluted with isolated dendritic cells (blue line), or cultured monocyte-derived dendritic cells as a positive control (green line), T cells: The starting ratio of the DC is 8:1. T cell proliferation was determined after 3 days by incorporation of 3H-thymidine.
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