Cell culture and reagents. Rat aortic endothelial
Lumacaftor (RAECs) were prepared as described previously [20]. RAECs were characterized by immunochemical staining using a monoclonal antibody (mAb, sc-59957, Santa Cruz, CA) for endothelial cells-specific VIII factor relative antigen. The endothelial cells were sub-cultured in medium-199 (Gibco BRL) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Invitrogen), penicillin (100 U/ml), and streptomycin (100 mg/ml) in a humidified CO2 incubator. RAECs from the third and fifth passages were used for all the experiments. Sub-confluent
cells were serum-starved for 24 h prior to the various treatments. Peripheral monocytes were isolated from healthy adult Wistar rats using a MACS cell separation kit (130-091-221, Miltenyi Biotec), and the purity was verified to be 95% by morphological examination after cytostaining using anti-rat CD14FITC mAb. The monocytes were maintained in RPMI-1640 with 10% FBS. All reagents were purchased from Sigma–Aldrich unless specified.
