Purification and identification of murine epidermal dendritic cells: flow cytometry and immunogold labeling.
We report on application of flow cytometric and immunogold labeling techniques to purify and identify two types of murine epidermal dendritic cells: Langerhans cells (LC) and Thy-1-positive dendritic epidermal cells (Thy 1+-dEC). After density centrifugation of epidermal cell (EC) suspensions through Ficoll gradients. IA-positive LC and Thy 1+-dEC are labeled with monoclonal antibodies (fluorescein-conjugated anti-IAd for LC and anti-Thy 1.2-biotin, followed by avidin-phycoerythrin, for Thy 1+-dEC). The fluorescence-activated cell sorter (FACS) is then used to obtain 95-98% pure populations of these dendritic cells with a yield of 2-4 X 10(6) cells and a viability of 80-90%. A post-fixation, pre-embedding immunogold labeling technique using 15 nm and 40 nm colloidal gold particles is employed to identify LC and Thy 1+-dEC, respectively, to confirm the purity of the sorting and to estimate the number of IA antigenic sites per LC. With transmission electron microscopy, ultrastructural morphology of sorted LC is preserved; however, Birbeck granules are markedly diminished compared to the pre-sorted population of LC. In contrast, characteristic dense-core granules are readily visualized in sorted Thy 1+-dEC. Purification of epidermal dendritic cells by flow cytometry may be a useful technique to employ in functional studies of epidermal dendritic cells.
D000818:Animals; D000954:Antigens, Surface / Q000032:analysis; D002469:Cell Separation; D003712:Dendrites / Q000648:ultrastructure; D000078404:Epidermal Cells; D004817:Epidermis / Q000276:immunology / Q000648:ultrastructure; D005260:Female; D005434:Flow Cytometry; D000949:Histocompatibility Antigens Class II / Q000032:analysis; D006651:Histocytochemistry; D007158:Immunologic Techniques; D007801:Langerhans Cells / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008854:Microscopy, Electron; D018800:Thy-1 Antigens
D000954:Antigens, Surface; D000949:Histocompatibility Antigens Class II; D018800:Thy-1 Antigens