@article{d0603435c91440bcb725131678ae7af3,
title = "FACS purification of bone marrow-derived epidermal populations in mice: Langerhans cells and Thy-1+ dendritic cells",
abstract = "A method was developed which allows for the separation and purification of Langerhans cells (LC) and Thy-1+ cells (Thy-1+dEC) from mouse epidermis. Epidermal cell (EC) suspensions were subjected to Ficoll separation, and the resulting interface EC were harvested. These EC were then 'tagged' with the appropriate monoclonal antibody and sorted into positive and negative populations using the Fluorescence Activated Cell Sorter (FACS). Preparations of viable LC and Thy1+dEC were obtained with 94-98% and 94-99% purities, respectively.",
author = "S. Sullivan and Bergstresser, {P. R.} and Tigelaar, {R. E.} and Streilein, {J. W.}",
note = "Funding Information: Mouse epidermis possesses at least 2 distinct populations of dendritic, bone marrow-derived cells: Langerh~ns cells (LC), and a recently observed population of cells wh1ch bears large amounts of the Thy-1 surface antigen (Thy-1+dEC) [1-4]. While the characterization of Thy-l+dEC is, as yet, in its infancy, there now exists a large body of experim~ntal data demonstrating that LC display important phenotypic markers and functional properties in common with antigen-presenting cells. LC bear cell surface Ia antigens in high density, Fe receptors, and C3b receptors, and they can act in a manner analogous to macrophages when test_ed in vitro _for th_eir cap~c ity to present antigen (5]. Strong Circumstantial ev1~ence mdicates that LC also perform critical antigen-presentmg function in vivo. Work from our own laboratories [6,7] has demonstrated that skin that is deficient in normally functioning LC fails to sustain the induction of contact hypersensitivity (CH) to a simple hapten. Moreover, im~unizing throu~h LCdeficient skin results in a state of spec1fic unresponsiveness which can be adoptively transferred with T lymphocytes to naive recipients (8). The generation of a positive im~unogen.ic signal in the presence of normal LC and unresponsiveness m Manuscript received August 31, 1984; accepted for publication December 3, 1984. Supported in part by grants CA-090B2 and AI-17363, and by a grant from Mary Kay Cosmetics, Inc. . * Portions of this work were presented at the Annual Meetmg of The Society for Investigative Dermatology, Inc., Washmgton, D.C., May 7-9, 1984. t Dr. Tigelaar is the recipient of N.I.H. Research Career Development Award AM-00940. :j: Current address: Department of Microbiology and Immunology, University of Miami School of Medicme, P.O. Box 016960, Miar:ni, Florida, 33101. Reprint requests to: Sabra Sullivan, Department of Dermatology, The University of Texas Health Science Center at Dallas, 5323 Harry Hines Boulevard, Dallas, Texas 75235. Abbreviations: CH: contact hypersensitivity EC: epidermal cell(s) MEM: Eagle's minimal essential medium F ACS: Fluorescence Activated Cell Sorter FCS: fetal calf serum LC: Langerhans cell(s) Thy-1+dEC: dendritic EC that bear large amounts of cell surface Thy-1 antigen their absence suggests that the epidermis may contain cellular elements capable of initiating both up-and down-regulating signals following the epicutaneous application of hapten. The possibility thus arises that LC themselves may be heterogeneous in this regard, and/or that another cell type within the epidermis contributes the down-regulating inf1uence. The discovery of a second population of bone marrow-derived, dendritic epidermal cells, Thy-1 +dEC, adds to the complexity of interpretations of earlier experiments, and it demands that purified populations of both Thy-1 +dEC and LC be obtained for study. This requirement to obtain purified preparations of epidermal LC has been perceived for some time, and investigators wishing to use LC for in vitro or in vivo experiments have utilized similar strategies to obtain them. In general, skin has been excised and exposed to trypsin; the epidermis has been removed and then utilized to prepare single cell suspensions of epidermal cells (EC). Efforts to enrich this resulting cell suspension for LC have included rosetting with antibody or complement-coated erythrocytes (9], the use of the Fluorescence Activated Cell Sorter (FAGS) (10], and {"}panning'' with an appropriate ligand bound to plastic dishes. Significant success in obtaining highly enriched preparations of LC has been reported for guinea pig and human tissues [11,12) while, by contrast, no such enrichment of LC has yet been reported for mouse skin. In this paper, we describe a protocol that allows us routinely to obtain from mice purified, viable epidermal LC and Thy-1 +dEC cells in numbers sufficient to conduct critical in vitro and in vivo experiments.",
year = "1985",
doi = "10.1111/1523-1747.ep12273454",
language = "English (US)",
volume = "84",
pages = "491--495",
journal = "Journal of Investigative Dermatology",
issn = "0022-202X",
publisher = "Nature Publishing Group",
number = "6",
}