The resulting supernatant, the initial water extract with no preservatives added, was stored at -20°C until needed. Before use, the extract was brought to room temperature and centrifuged at 18,000 rpm (38,700 g) for 20 min, and the pellet was discarded. The supernatant was then passed through a 0.2 micron Acrodisc (Gelman Science, USA) syringe-adapted filter. This procedure removed much of the high molecular weight complex material, which consisted mainly of membrane vesicles and intact flagellae.
HUVEC were harvested from umbilical cords by collagenase treatment as previously described . The cells were plated in Medium 199 (Gibco, USA) supplemented with 10% heat-inactivated fetal calf serum (JRH Biosciences, USA), thymidine (2.4 mg/L; Sigma Chemical Co, USA), glutamine (23 mg/L; Gibco, USA), heparin sodium (10 IU/mL; Mochida Pharmaceutical Co Ltd, Japan), penicillin G/streptomycin/Fungizone (Gibco, USA) and endothelial cell supplement: mitogen (80 mg/mL; Biomedical Technologies Inc, USA). The cell cultures were incubated at 37°C in a humidified atmosphere with 5% CO2 and expanded by brief trypsinization (0.25% trypsin in phosphate buffered saline (PBS) containing 0.02% EDTA). First through third passage HUVEC were seeded into gelatin-coated (0.1%), 96-well tissue culture plates and used when confluent.
The monoclonal antibodies (mAbs) directed against ICAM-1 (LB-2) , VCAM-1 (E1/6) , and E-selectin (H18/7) were supplied by Becton Dickinson, USA. These antibodies reportedly block the adhesive reactions of leukocytes and endothelial cells under static conditions.