Endothelial cell wound healing assay

HMEC-1 micro-endothelial cells were grown to confluence on 24-well plates. Then, 18 hours before starting the assay, HMEC-1 cells were subjected to FBS starvation to avoid proliferation effects. Consistently shaped wounds were made using a sterile 100-µl pipette tip across each well, creating a cell-free area line [70]. At that point, cells were exposed to the supernatants of EL4, K12 or MDA-MB-231 cells (previously treated with LMW HA, DOX, or LMW HA + DOX) diluted at 1:1 ratio with DMEM. For negative control, cells were exposed to DMEM without FBS. Controls to discard residual effects of DOX or HA on ECs were also performed. Three images were captured in the same coordinates point at 0, 4, 8 and 22 h after performing the wound. The gap size of the wound was measured and analyzed using ImageJ 1.50b software package.