,
,
, Belgium) were cultured in endothelial cell basal medium (ECBM, PromoCell, Heidelberg, Germany) supplemented with 10% fetal calf serum (FCS), 4 ?L/ mL endothelial cell growth supplement/heparin, 0.1 ng/mL human epithelial growth factor, 1 ng/mL human basic fibroblast growth factor, 1 ?g/mL hydrocortisone (C-39210, PromoCell), 2 mM glutamine, 100 U/mL penicillin, and 100 ?g/mL streptomycin (Life Technologies, Biological Experimental Procedures. Endothelial Cell Culture and Activation. Primary human umbilical vein endothelial cells (HUVECs, Lonza Verviers SPRL
Controls were performed using diluent (DMSO 1/ 1000) alone. ZA was used as a control ,
, Screening by Cellular ELISA. HUVECs were plated in 96-well plates (1 × 10 4 cells/well) and cultured overnight to reach confluency
After incubation with the substrate, the optical density was analyzed at 405 nm. Extracts and pure compounds were tested at concentrations of 5, 25, and 50 ?g/mL and ?M, respectively. Experiments were performed in triplicates and repeated in three independent assays. Results were expressed as percentage of inhibition compared to treatment with TNF (VCAM-1) or IFN? (MHC molecules) alone. Immunostaining and Flow Cytometry. Confluent HUVEC monolayers were incubated for 1 h with 3 at a final concentration of 10 ?M, negative control (DMSO dilution 1/1000 in culture medium), or positive control (ZA; 10 ?M) before addition of IFN? (100 U/mL for 48 h), and MICA proteins were detected using a specific mouse monoclonal antibody (5 ?g/mL, R&D Systems) and revealed using horseradish peroxidase-labeled secondary antibodies (1 ?g/mL, Cell Sciences ,
, , pp.0-0001
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,
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