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Purification and identification of murine epidermal dendritic cells: flow cytometry and immunogold labeling

CA Picut, CS Lee, EP Dougherty, KL Andersen and RM Lewis

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.

Volume 35, Issue 7, pp. 745-753, 07/01/1987
Copyright © 1987 by The Histochemical Society


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