Recombinant human vascular endothelial growth factor

Cells were seeded in 24-well Matrigel-coated plates and were treated with samples as is described below. The capillary-like tubular formations were considered for evaluating angiogenic or antiangiogenic effects of the test sample of endothelial cells on Matrigel base (Invitrogen, USA) as described before [my article]. Matrigel thawed on ice was added 100 μl/well of a 24 well plate, carefully and incubated for 30 min in 37°C allowing matrigel to form a jelly base with a flat surface. About 40 μl of HUVECs suspension (10⁵ cells/well) were seeded in each well. The wells were treated with 40 μl of test samples with the final concentrations of 5, 50, and 500 μg/ml per well at the same time. Blank or 10 ng/ml recombinant human vascular endothelial growth factor (VEGF) (R and D Systems, USA) alone served as a negative or positive control and culture medium added up to 300 μl. The plate was incubated for 24 h in 37°C and after incubation in vitro endothelial tube formation data were expressed as a percentage of the number of capillary-like tubes in treating wells to untreated control wells of triplicate readings.[4 8 (link)] Capillary-like tube formation was discovered with a Nikon inverted microscope and the images recorded digitally using Angioquant analysis software (Tampere University of Technology, Tampere, Finland).[9 (link)]