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BLAST searches of the National Center for Biotechnology Information expressed sequence tag (EST) database for mouse chemokine sequences identified one mouse EST (AA175762) that encoded a 92-amino acid protein with 64% identity to human MDC. This sequence is identical to the recently described mouse ABCD1 sequence (8). An 800-base pair Eco RI fragment containing the coding region was used as a probe for Northern blot analysis, and digoxigenin-based In situ hybridization was performed as described (4). RT-PCR was performed with primers specific for MDC (5′-GTGGCTCTCGTCCTTCTTGC-3′ and 5′-GGACAGTTTATGGAGTAGCTT-3′). ELC (4), or actin. Mature mouse MDC (amino acids 25 to 92) was expressed in Escherichia coli as described for ELC (4).
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Skin was sensitized as described (17). Mice shown in Fig. 2A were painted on the shaved thorax and abdomen with 0.4 ml of 2% FITC (Sigma) dissolved in a 1:1 (v/v) acetone/dibutyl phthalate (Sigma) mixture, and RNA was prepared from pooled brachial, inguinal, and axillary nodes. For other experiments, a region of mouse skin on the arm or flank was shaved, 25 μl of 1% FITC in acetone/dibutyl phthalate was applied onto each region, and the draining nodes (brachial and inguinal) were analyzed 1 day later.
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6 cells/ml for 1 day. The stromal material that did not enter suspension after mechanical mashing contained SLC-expressing cells and ELC-expressing resident DCs (4, 14). Stromal preparations from 30 to 40 brachial and axillary nodes per experiment were cultured overnight in 2 ml of serum-free medium to provide supernatant for chemotaxis assays.
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0345138732
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We thank R. Locksley, S. Luther, K. Reif, and A. Weiss for comments on the manuscript; M. Ansel for help with the in vivo transfer experiments; and C. McArthur for cell sorting. Supported in part by NIH grant AI-40098, the Pew Foundation (J.G.C.), and the American Lung Association (H.L.T.).
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