*4.5. Cell Migration*

First, 0.6 mL medium containing 10% FBS was added to the lower chamber of Transwell chambers (6.5 mm diameter, 8 μm pore size; Corning Life Sciences), and cells suspended in a serum-free medium at a density of 1.5 × 10<sup>5</sup> cells/mL were seeded (0.1 mL) in the upper chambers. Then, 400 μg/mL PSS was added to both the upper and lower chambers. After incubation for 16 h, the cells were fixed by cold 4% paraformaldehyde, stained by 0.1% crystal violet, and cells that had not migrated were removed from the upper chambers. The remaining cells were photographed in five random fields per membrane. The dye was dissolved in 80 μL of acetic acid, and the absorbance of the resulting solution was measured at 600 nm using a microplate reader (SpetraMAX i3, Molecular Devices, Sunnyvale, CA, USA).

### *4.6. The Wound Healing Assay*

The e ffect of PSS on migration was analyzed in vitro using a wound healing assay. B16-F10 cells were seeded in 12-well culture plates to reach 70% confluency. The cell monolayer was scratched vertically down the center of each well with a sterile 200 μL micropipette tip, and rinsed carefully with phosphate bu ffer solution (PBS) three times to remove cell debris. FBS-free medium with varying concentrations of PSS (100, 400 μg/mL) or 400 μg/mL heparin was added to each well. Three randomly selected views along the wound line in each well were photographed under an inverted microscope at 0 h and 24 h after incubation. The percentage of void area with respect to time 0 was determined using ImageJ software (ImageJ 1.8.0, Rawak Software Inc., Stuttgart, Germany).
