2.2.7. Exclusion Assay

HUVEC pericellular coat was visualized and measured by using a particle exclusion assay. In total, 6 × 1000 cells/well were seeded in 12-well plate and treated with TNF-α or PBS as control. After 24 h, 500 µL of a suspension of formaldehyde-fixed erythrocytes (15 × 1,000,000 erythrocytes/mL) was added to the wells and allowed to settle for 20 min at 37 ◦C. Images of the pericellular coat were obtained using phase contrast microscope Olympus IX51. The presence of HA on the pericellular coat was evaluated treating the cultures with 2 U/mL of Hyaluronate Lyase for 1 h at 37 ◦C before visualization with the particle exclusion assay. Representative cells were photographed at a magnification of ×40; the control experiment was performed with heat inactivated Hyaluronate lyase. ImageJ software was used to quantify the area delimited by red blood cells and the area delimited by the cell membrane to give a coat-to-cell ratio [25].
