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3, at pH 7.4)]. Subsequently, the cells were directly stained with fluorescein isothiocyanate-or phycoerythrin-conjugated mouse monoclonal antibodies to CD83 (Immunotech, Marseille, France), CD1a, CD86, HLA-DR (PharMingen, San Diego, CA), or CCR6 (R&D Systems) at 24°C for 1 hour at a final concentration of 5 μg/ml in FACS buffer. As a negative control, DCs were stained with isotype-matched irrelevant antibodies. Thereafter, the cells were washed twice with FACS buffer and twice with PBS, resuspended in PBS containing 1% paraformaldehyde, and analyzed with a flow cytometer (Coulter or FACScan Epics Analyzer).
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85069096391
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Parental HEK293 epithelial cells were transfected with linearized CCR6 mammalian expression constructs by electroporation. After selection in Dulbecco's minimum essential medium (Biowhittaker), 10% FBS, 2 mM glutamine, penicillin (100 U/ml), and streptomycin (100 μg/ml) containing G418 (400 μg/ ml) (Life Technologies, Gaithersburg, MD) for 2 weeks, single-cell cloning was performed. The expression of functional CCR6 was confirmed by saturation binding, FACS analysis of surface CCR6, and chemotaxis of the transfectant cells in response to LARC.
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Data available at www.sciencemag.org/feature/data/ 1042876.shl.
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note
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We thank P. W. Gray and M. Tsang for human CCR6 expression constructs and antibody to CCR5, respectively; N. Dunlop for technical assistance; and C. Fogle and C. Nolan for secretarial assistance. Supported in part by a fellowship from the Office of International Affairs of the National Cancer Institute (NCI) of NIH (to D.Y.) and by contract N01-CO-56000 from NCI of NIH (to O.C. and O.M.Z.H). The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. government.
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