Mouse anti vcam 1 monoclonal antibody

Expression of VCAM-1 on the surface of HUVECs was evaluated by incubating 2 × 10⁵ HUVECs in EGM in the absence or presence of 20 ng ml⁻¹ human TNFα (hTNFα Millipore, Billerica, MA, USA) for 24 h. After harvesting and washing with phosphate-buffered saline solution (PBS), the cells were fixed in 4% paraformaldehyde (PFA) for 20 min at room temperature. After blocking with PBS containing 1% bovine serum albumin (BSA) for 1 h at room temperature, the cells were incubated first with mouse anti-VCAM-1 monoclonal antibody (1 μg ml⁻¹; Abcam, Cambridge, UK) for 1 h at 37 °C, and then with Alexa Fluor 488-conjugated anti-mouse antibody (1:1000; Invitrogen) for 1 h at 37 °C. The effects of anti-VCAM-1-D6 IgG on endothelial cell activation were evaluated by incubating 2 × 10⁵ HUVECs in the absence or presence of 20 ng ml⁻¹ hTNFα (Millipore), 20 μg ml⁻¹ control IgG or anti-VCAM-1-D6 IgG for 24 h. After blocking with PBS containing 1% BSA for 1 h at room temperature, the cells were incubated first with rabbit anti-ICAM-1 monoclonal antibody (1:500; Abcam) for 1 h at 37 °C, and then with an Alexa Fluor 488-conjugated anti-rabbit antibody (1:1000; Invitrogen) for 1 h at 37 °C. Samples were analyzed by flow cytometry using a FACSCalibur system (BD Biosciences, San Jose, CA, USA) with the aid of FlowJo software (TreeStar, Ashland, OR, USA).