Column-free isolation of untouched mouse plasmacytoid dendritic cells (P4149)

Abstract

Abstract Plasmacytoid dendritic cells (pDC) are a DC subset distinct from conventional dendritic cells (cDC) in that they are able to produce large amounts of type I interferon (IFN) after challenge with pathogens. Viruses activate pDC mainly through Toll-like receptors (TLR). Unlike cDC, pDC are poor antigen-presenting cells. In line with the functional differences, pDC express lower levels of CD11c and MHCII as compared to cDC. Moreover, a number of markers expressed by pDC are absent on cDC including: PDCA-1, Siglec-H, B220 and Ly-6C. pDC are found in both lymphoid and non-lymphoid organs. Here, we describe a negative selection method to isolate untouched pDC from mouse spleen. This method uses an immunomagnetic, column-free cell separation technology (EasySepTM). Briefly, single cell suspensions of splenocytes are labeled with biotinylated antibodies against non-pDC. Bi-specific antibody complexes against biotin and dextran are used to cross-link non-pDC with dextran-coated magnetic particles. The unwanted cells are then removed using an EasySepTM magnet. The procedure can be automated using RoboSepTM. Starting with 0.4±0.2% CD11c+PDCA-1+ pDC in spleen, purities of 79±9% (n=21) are achieved. The isolated pDC are functional and produce IFN-α in response to TLR9 ligand, ODN-1585 as measured in ELISA. pDC play an important role in anti-viral immunity and autoimmunity, therefore the isolation of untouched pDC is critical in studies examining their role in these immune responses.