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2. Nonadherent cells were removed, and complete medium (RPMI 1640 containing heat-inactivated newborn calf serum) was added. After incubation of macrophage monolayers for 20 hours, tyrphostins were added at a final concentration of 20 μM followed 2 hours later by the addition of E. coli 055:B5 LPS (10 μg/ml). Endotoxin (Sigma) was prepared by phenol extraction. After incubation for 6 hours, supernatants were collected and the concentration of TNF-α was determined with an ELISA kit (Endogen). The results are expressed as the mean of two determinators; deviations from the mean did not exceed 8%.
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- production seems to depend on the method of macrophage preparation, whereas maximal LPS does not depend on the preparation.
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84901964434
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The tyrphostins AG 490 and AG 556 were found to be more active in inhibiting TNF-α cytotoxicity in vitro (two- to threefold). This finding suggests that different sets of PTKs mediate the effects of LPS and TNF-α and, therefore, that different families of tyrphostins will be effective against these two agents.
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6 U/mg (Reportech, Rocky Hill, NJ) at 0.0, 0.2, and 0.5 ng/ml with and without 10 μM tyrphostin. After incubation for 18 hours, the supernatants were aspirated, the monolayers were washed twice with PBS, and 200 μl of neutral red solution (0.02%) was added. After incubation for 2 hours, cells were washed out and the dye that had been absorbed by the live cells was extracted upon the addition of 200 μl of Sorenson buffer containing 50% ethanol. The concentration of the dye was determined by an ELISA autoreader with a 550-nm filter.
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note
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We thank M. Collazo from SUGEN, Inc. (Redwood City, CA) for helping in the preparation of this manuscript.
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