24 well transwell polycarbonate filters

Cell migration assays were done in 24-well transwell polycarbonate filters (pore size, 8 mm; Corning Costar). Briefly, LX-2 cells were seeded in the upper chamber, and chemoattractants such as Gal-1, PDGF, and TGF-β were added to the lower chamber. Migrated cells were counted after cells had been individually incubated for 24 h. Non-penetrating cells were removed from the upper surface of the filter with a cotton swab. Penetrating cells were fixed and stained with a DiffQuick stain kit (Dade Behring) according to the manufacturer’s instructions. For quantification, all of the cells that had migrated onto the lower surface were stained and counted under a light microscope. All experiments were performed in duplicate. Results are shown as the mean ± standard error of the mean (SEM) of three independent assays.

Equal numbers of cells were plated in 6-well plates; wounds were generated when the cells reached 90% to 95% confluency with a sterile pipette. Wounds were photographed at various time points with a phase-contrast microscope (Olymbas). A Transwell migration was assessed using 24-well Transwell polycarbonate filters (Corning Costar Corp.) with an 8-um pore size. Approximately 50,000 cells were seeded onto each upper chamber of 24-well Transwell plates and incubated in serum-free medium. Medium containing 10% FBS was placed in the bottom chamber and served as a chemoattractant. Eight or twelve hours later, cells on the upper surface of the filter were removed by gently wiping with a cotton swab. Cells that migrated to the bottom of the filter were fixed and stained with crystal violet. Migrated cells were visualized by microscope and counted in at least five random fields.