Thrombin-induced surface expression of DAF requires de novo protein synthesis.

HUVECS were plated at confluence (5 × 10⁵ cells/well) in 6-well dishes and cultured overnight at 37°C. They were then pretreated with CHX (1 μg/mL) for 30 minutes prior to addition of thrombin (10 U/mL) for a further 24 hours. Following harvesting, DAF expression was measured by flow cytometry using MoAb 1H4. CHX at these concentrations was not toxic to ECs, as assessed by examination of the monolayers prior to staining using phase-contrast microscopy, cell counting, and estimation of trypan blue exclusion. The data are expressed as the RFI ± SEM from 2 similar experiments performed on separate HUVEC cultures.