Culture inserts for live cell analysis

Cell migration was determined by transwell assay and wound healing assay. For transwell assay, 3×10⁴ cells were seeded on collagen (1/100, Millipore) coated insert (0.8μm, BD Bioscience) of 6 well-plates in RPMI containing 10% FBS or 0.1 % FBS in the presence of HGF. At the end of the experiments, cells migrating to the other side of the filter were stained with 0.5% crystal violet and counted. Wound healing test was performed by using Culture-inserts for Live Cell Analysis (Ibidi). A total of 3×10⁴ cells were plated on each compartment of insert and cultured for 5h. The insert was then removed (0h) and cells were further cultured for 24h. Cells were photographed at 0h and after 24h of culture to record the wound width.

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%.

Wound healing assay was performed with the assistance of Culture-Inserts for live cell analysis (Ibidi, Martinsried, Germany). 70μl/well of cell suspensions were seeded into the culture inserts at a concentration of 3×105/ml. Following culture overnight, culture inserts were removed leaving a gap of 500μm between the cell population. Followed by PBS washing to remove cell debris, the cells were cultured in fresh medium and the growth pattern was photographed using a microscope within 48 hours.