Transwell 24 well boyden chamber with a 8 m polycarbonate membrane

Cell migration was assayed using the Transwell 24-well Boyden chamber with a 8-µm polycarbonate membrane (Corning, Inc.). Briefly, 3.0×10⁴ cells were plated in the upper chamber containing 200 µl serum-free media, while the bottom chamber contained 500 µl media supplemented with 10% FBS as a chemoattractant. After 48 h, the migrated cells were fixed by 4% paraformaldehyde, stained with 0.1% crystal violet, and finally eluted with 1 ml of 33% acetic acid. Absorbance of the migrated cells was measured at 460 nm and expressed as an A460 value. Three independent experiments were performed.

A Transwell 24-well Boyden chamber with a 8-µm polycarbonate membrane (Corning, Inc.) was used to examine the migration capacity. For invasion assay, the Transwell insert was pretreated with Matrigel (BD Biocoat) at 37°C for 8 h. LCC cells (1×10⁵ cells) were seeded in serum-free medium in the upper chamber of the insert; medium with 20% FBS was included in the lower chamber. After 24 h, cells on the bottom surface of the filter were fixed with 4% polyoxymethylene at room temperature for 15 min and then 1% crystal violet dye was used to stain the cells at room temperature for 10 min. Cells were visualized using a Leica AF6000 LX fluorescence microscope (Meyer Instruments, Inc.) and counted in 5 randomly selected fields of vision at a magnification, ×100.