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3CN system. The derivative was homogeneous on HPLC and by SDS acrylamide electrophoresis. It gave a relative molecular mass of 7901.47 ± 0.58 on electrospray mass spectrometry (calculated, 7901.03).
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Monocyte chemotaxis was carried out as described [M. Lusti-Narasimhan et al., J. Biol. Chem. 270, 2716 (1995)] with the following modification. The monocytes were purified from the white cell fraction in blood (buffy coats) diluted with 100 ml of phosphate-buffered saline (PBS) before loading onto Ficoll for separation of lymphocytes.
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note
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125I.
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13
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1842409316
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note
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50, respectively, and reincubated at 37°C for 3 hours. Input virus was then washed out before adding growth medium containing appropriate chemokine concentrations. The cultures were incubated at 37°C for up to 12 days with medium changes twice weekly but without further addition of chemokine. Virus production into the supernatant was assessed by measurement of RT activity using a sensitive nonradioactive method (Retrosys RT activity kit; Innovagen AB, Lund, Sweden).
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10344266976
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1842416880
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note
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8 cells in 20 ml of RPMI 1640 were added to a bacterial culture dish (diameter 140 mm). After 2 hours of incubation, cells that had not adhered were washed away and RPMI 1640 plus 10% human serum was added. After overnight incubation, loosely attached cells were again washed away, RPMI 1640 containing 20% human serum was added, and the cultures were incubated for another 3 days. The day before infection with HIV, cells were treated with versene, gently scraped off using a cell scraper, and reseeded into cell culture dishes. At this stage, >99% of cells expressed the macrophage marker CD14. Chemokine treatment and HIV infection were carried out as described for PBMC cultures (12). Culture supematants were tested for RT activity after 3 weeks.
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19
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1842368829
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note
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50) was inhibited by RANTES but was only slightly affected by Met-RANTES. AOP-RANTES inhibited macrophage infection by each of the four strains.
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21
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0026656502
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50 and incubated for a further 3 hours. The cells were washed in growth medium and incubated for 4 days in medium containing the appropriate chemokine before fixing and staining in situ for p24 antigen as described [P. R. Clapham, Á. McKnight, R. A. Weiss, J. Virol. 66, 3531 (1992)]. Numbers of stained cells were estimated by microscopy.
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Clapham, P.R.1
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Endres, M.J.1
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1842297903
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note
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We thank Á. McKnight, M. Dittmar, C. A. Power, M. Luther, M. Marsh, and R. A. Weiss for helpful discussions, J. D. Reeves for help and advice with transfections, and B. Dufour and F. Borlat for excellent assistance. Primary HIV-1 strains (13) were provided by S. Beddows, J. Weber, G. Carnegie, U. Desselberger, and the UK HIV Molecular Epidemiology Group. Technical facilities in R.E.O.'s laboratory were financed by the Swiss National Science Foundation. Supported by the Medical Research Council, UK.
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