Matrigel growth factor reduced basement membrane matrix

Human CAECs were seeded on the top of Matrigel (Corning® Matrigel® Growth Factor Reduced Basement Membrane Matrix, cat# 356231) either in monoculture (4000 cells/well) or in coculture with PCs (4000 CAECs + 1500 PC/well), using Angiogenesis μ-Slides (IBIDI, U.K.) and growth factors-free medium. The S protein (1 μg/ml – 5.8 nM) or PBS vehicle were added to the system. Images were taken after 5 h using an inverted Leica microscope equipped with a 5× objective. The total tube length per imaging field was measured using ImageJ. To assess the interaction between PCs and CAECs, PCs were labelled with the red fluorescent tracker Vybrant™ DiI Cell-Labeling Solution (Invitrogen; dilution 1:1000 in PBS, incubation for 5 min at 37°C followed by 15 min at 4°C). For experiments requiring the CD147 blockade, 100000 PCs in a total volume of 100 μl were pre-incubated with the anti-CD147 antibody (20 μg/ml) for 1 h at 20°C. Per each patient, experiments were performed in triplicates.