Measuring Cell Migration in Wound Healing Assay

The wound healing assay or scratch test was performed as previously reported [25 (link)]. Briefly, 6 × 10³ U87-MG cells were seeded in multiwell-12 plates containing culture inserts for live cell analysis (Ibidi, Gmbh, Martinsried, Germany) and grown to confluence in DMEM/10% FBS for 24 h. After removing the inserts, the cells were pre-treated for 40 min at 37 °C with uPAcyclin or diluents at the indicated concentrations, and then incubated in DMEM/3% FBS for 24 h. The images were taken using the Inverted Microscope Leica DMI6000 DFC 402 (Leica Microsystems, Milan, Italy). A total of 10 × 10⁴ C6 cells were grown to confluence in 12-well plates, in the presence of DMEM/10% FBS. Then, the cell monolayers were wounded with a yellow tip, pre-incubated with uPAcyclin (10 pM and 100 nM) or diluents, and incubated in DMEM/2.5% FBS for 16 h. The images were obtained using the Inverted Axiovert 25 Microscope, equipped with the Zen 3.3 Software. To quantitate cell migration, a rectangle covering the wound edges is drawn on the T0 image and is further applied to the single photograms of each sample. The average margin distance is measured at three points and the results are expressed as percentage wound width of the T0 distance, taken as 100%.