Endothelial Tube Disruption Assay

HUVECs (Invitrogen, Waltham, MA USA) were plated at a high concentration (124,000 cells/well) on 24-well culture plates (Corning Life Sciences, Glendale, AZ, USA) that had been coated with Matrigel^® (300 μL/well, 9.5 mg/mL, BD Pharmingen, San Diego, CA, USA) and maintained at 37 °C for 16 h in Human Large Vessel Endothelial Cell Basal Medium (formerly M200, Gibco/ThermoFisher Scientific, Waltham, MA, MA), supplemented with a high growth factor supplement kit (ATCC). The plates were checked to confirm a two dimensional capillary-like network had formed, and tube disruption was induced by the addition of varying concentrations of OXi6196 or colchicine (10 nM, 100 nM, and 1000 nM) for 2 h. Medium was removed and the cells were washed twice with M200. The plates were photographed (nine fields per well at 5× magnification) using a Canon Powershot A640 digital camera mounted onto an Axiovert 40 CFL inverted microscope (Zeiss, Thornwood, NY, USA) [31 (link)].