2.9. Transendothelial Migration Assay

One × 10⁵ HPMEC cells were plated in the upper chamber of a collagen-coated transwell insert with 8 µm pore size (Corning Costar, Cambridge, MA, USA) and grown in the complete endothelial medium for 4 days to reach 100% confluence. Melanoma cells were pulsed with 25 µM CellTracker Green CMFDA (Invitrogen, Waltham, MA, USA) dye for 30 min before being trypsinised and plated on top of the HPMEC monolayer. Cells were allowed to migrate for 24 h toward the complete DMEM in the lower chamber of the transwell. Transwell inserts were fixed with 1% PFA and permeabilised by 0.5% Triton X-100. Non-migrated cancer cells were removed from the upper side of the filter using cotton buds. Migrated cells on the lower side of the filter were visualised under a Nikon Ti-E fluorescence microscope (Nikon, Tokyo, Japan). Five fields from each insert were captured and quantified using the Image J software.