| Overview |
The CD304 (BDCA-4/Neuropilin-1) MicroBead Kit has been developed for the enrichment of plasmacytoid dendritic cells (PDCs) from PBMCs by positive selection.
The kit includes FcR Blocking Reagent and CD304 (BDCA-4/Neuropilin-1) MicroBeads. |
| Details |
Background information
CD304 (BDCA-4/Neuropilin-1)1 is specifically expressed by PDCs in human blood1–5, bone marrow6, and cord blood11. Exclusive expression of CD304 (BDCA-4/Neuropilin-1) on PDCs allows their direct isolation. In blood and bone marrow, CD304 (BDCA-4/Neuropilin-1)+ PDCs are CD4+, CD45RA+, CD303 (BDCA-2)+, CD141 (BDCA-3)dim, CD1c (BDCA-1)–, and CD2–. They lack expression of lineage markers (CD3, CD14, CD16, CD19, CD20, CD56) and neither express myeloid markers such as CD13 and CD33, nor Fc receptors such as CD32, CD64, or FceRI. Their lymphoid origin is indicated by their plasmacytoid morphology (fig. 2) and the expression of the pre-T cell receptor α-chain. Freshly isolated CD1c (BDCA-1)+ or CD141 (BDCA-3)++ myeloid blood dendritic cells and monocytes do not express CD304 (BDCA-4/Neuropilin-1), but expression of CD304 (BDCA-4/Neuropilin-1) is induced on myeloid blood dendritic cells and monocytes upon culturing.1, 6 In inflamed tonsils, CD304 (BDCA-4/Neuropilin-1) expression is, apart from PDCs, also detected on some other cells, primarily follicular B helper memory T cells.6
CD304 (BDCA-4) was shown to be identical to neuropilin-1 (NP-1).6 Neuropilin-1 has formerly been discovered to be expressed on numerous nonhematopoietic cell types including neurons, endothelial and tumor cells.
Unlike binding of antibodies to CD303 (BDCA-2), binding of antibodies to CD304 (BDCA-4/Neuropilin-1) does not have a substantial effect on IFN type I production in PDCs after induction by, e.g., influenza virus (fig. 3).5, 6, 14
Downstream applications
Isolated CD304 (BDCA-4/Neuropilin-1)+ PDCs were used, for example, to examine expression of Toll-like receptors4, 7, 8, 12, chemokine receptors3, 8, 10, or new antigens, e.g., EMR2,9 and for studies on dendritic cell activation4, migration3, cytokine production4, 6, 8, and T cell polarization4, 6, 13. Functional and phenotypical analysis was performed, e.g., of PDCs isolated from PMBCs and synovial fluid from psoriatic arthritis and rheumotoid arthritis patients15. |
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| Figure 1 |
| Isolation of CD304 (BDCA-4/Neuropilin-1)+ plasmacytoid dendritic cells from PBMCs using the CD304 (BDCA-4/Neuropilin-1) MicroBead Kit, MS Columns, and a MiniMACS™ Separator. Cells were stained with CD303 (BDCA-2)-FITC and CD123-APC. Like CD304 (BDCA-4/Neuropilin-1), the CD303 (BDCA-2) antigen is specifically expressed on plasmacytoid dendritic cells in blood, and allows their direct identification. Apart from plasmacytoid dendritic cells, CD123 is also expressed at high levels on basophils and at low levels on monocytes and myeloid dendritic cells. |
| PBMCs before separation |
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| Enriched CD304 (BDCA-4/Neuropilin-1)+ plasmacytoid blood dendritic cells |
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| Figure 2 |
| Plasmacytoid dendritic cells were isolated from PBMCs by using the CD304 (BDCA-4/Neuropilin-1) MicroBead Kit, and May-Grünwald/Giemsa stained. |
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| Figure 3 |
| IFN-α secretion of CD304 (BDCA-4/Neuropilin-1)+ blood dendritic cells. Plasmacytoid dendritic cells were isolated using the CD304 (BDCA-4/Neuropilin-1) MicroBead Kit and cultured for 24 hours in the presence of 10 ng/mL IL-3 with and without influenza virus (5 HAU/mL, strain PR8). Secretion of IFN-α was determined by ELISA.5,6 |
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