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LCs were extracted from normal human skin. Epidermal suspension and enrichment of LCs followed the protocol of Teunissen et al. (13), with some modifications [H.-N. Liu, Y.-T Chang, C -K. Wong, Clin. Exp. Dermatol. 19, 113 (1994)]. Skin tissue was digested overnight at 4°C with 2 mg/ml of dispase in RPMI media with 20% fetal calf serum (FCS) and 1% antibiotics. The epidermis was separated and incubated for 15 min with trypsin (0.005%) and deoxyribonuclease (100 U/ml) in phosphate-buffered saline (PBS). The cell suspension was loaded on a discontinuous Ficoll-Metrizoate gradient LCs were recovered from two interfaces, one between the sample and the gradient and the second between layers with densities of 1 068 and 1 057 g/ml. Purity was estimated with immunofluorescence staining with CD1a [OKT6 (Ortho)], CD3, and CD14. The cells were maintained in RPMI media with 20% FCS and 1% antibiotics. No clumping of LCs with keratinocytes was observed. The viability according to Trypan Blue exclusion was higher than 85% for the period of observation in the assay.
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PBMCs from HIV-1-seronegative individuals were purified by the Ficoll-Hypaque method, stimulated with 10 μg/ml of phytohemagglutinin A (PHA) (Sigma) for 72 hours, and maintained in RPMI 1640 with 20% fetal bovine serum, 10 U/ml of recombinant human interleukin-2, 100 U/ml of penicillin, and 100 μg/ml of streptomycin.
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21
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note
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6 PBMCs per cubic centimeter and RPMI 1640 plus 5% (v/v) human serum and 20% fetal bovine serum. After 5 days, cells were washed twice with PBS and maintained with RPMI 1640 with 20% fetal bovine serum, 100 U/ml of penicillin, and 100 μg/ml of streptomycin.
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note
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6 cells/ml in the same media as for MDMs and PMBCs.
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23
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13344257608
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note
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6 MT-2 cells were challenged with 1 ng/ml of p24 from each isolate on day 1. All the LC assays were done 24 hours after the extraction and enrichment of the cells Assays with MDMs were started 4 days after the removal of nonadherent cells. Cell cultures were inoculated with each isolate for 18 hours at 37°C, washed with PBS, and resuspended in fresh complete media. Infections with PBMCs, MDMs, and MT-2 cells were done twice, and LC infections were done in duplicate with at least two different LC preparations To determine p24 levels, we obtained culture supematants by removing cells and cell debris after centrifugation at 3500 rpm for 10 min from LCs, PBMCs, and MDMs on days 7, 14, and 21. Standard dilutions were done in each sample, and the procedure was performed according to the manufacturer's protocol (NEN-Dupont Core profile enzymelinked immunosorbent assay).
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13344264502
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note
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We acknowledge support of the research staff of the Fenway Community Health Center, especially R. Goldstein. This research was supported in part by grants CA 39805 and HL 33774 from NIH and by a grant from the Massachusetts Department of Public Health L.E S.-R, R S., C.A., and P.A were supported by training grant 5 D43 TW0004 from the Fogarty International Center, NIH. The authors thank K. MacArthur for editorial assistance.
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