The Isolation of Mouse Dendritic Cells from Lymphoid Tissues and the Identification of Dendritic Cell Subtypes by Multiparameter Flow Cytometry

Dendritic cells (DCs) are powerful initiators of the adaptive immune system and their manipulation is seen as a viable approach in the treatment of many human ailments. Our isolation method purifies DCs from mouse lymphoid organs by efficiently removing them from the tissue using collagenase, selecting the light density fraction of cells and then negatively selecting for DCs using a combination of monoclonal antibodies directed against non-DC lineage cells together with anti-rat immunoglobulin-coated magnetic beads. Remaining contaminating cells can be depleted using a combination of specific biotinylated antibodies and anti-biotin-coated magnetic beads, in the case of natural killer (NK) cells, or by FACS gating, during sorting or analysis, in the case of autofluorescent macrophages. The heterogeneous DCs can then be separated into various functionally different populations via immunofluorescent labelling and cell sorting. The study of mouse DC populations may enable us to align them to their human counterparts and subsequently to DC populations produced in vitro.