2 7 bis 2 carboxyethyl 5 and 6 carboxyfluorescein acetoxymethyl ester bcecf am

Cell-cell adhesion was performed as previously described (8 (link), 32 (link)–35 (link)). Confluent HUVEC monolayers were used and MNC adhesion to them was assessed. HUVEC monolayers were stimulated, where indicated, with TNF (10 ng/ml) for 24 h. In the experiments with siRNA-mediated knockdown of Del-1 in HUVEC, endothelial cells were transfected with control siRNA or Del-1 siRNA 48 h prior to the experiments. MNC were stained with Cell Tracker Green-CMFDA (Molecular Probes, Eugene, Oregon) or with 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) (Molecular Probes, Eugene, Oregon) and after detachment with trypsin, cells were resuspended in RPMI 1640 with 0.05 % bovine serum albumin. A total of 100,000 cells MNC/well (in 100 μL RPMI 1640 with 0.05 % albumin) was added to the HUVEC monolayers and after incubation for 20 minutes at 37°C, the plates were washed with warm RPMI 1640 to remove non-adherent cells. Adherent cells were quantified in triplicates with a fluorescence plate reader (Synergy HT, Biotek, Germany).