24 well plate transwell chamber

The invasion assay was performed using a 24-well plate transwell chamber containing 8-μm wells (Corning, USA). The top chamber contained Matrigel (BD Biosciences). Cells (1 × 10⁵/well) were inoculated in a serum-free medium in the top chamber, while the bottom chamber contained the medium, along with 10% FBS. The invasion of cells was allowed to occur for 48 h at 37°C, after which they were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet solution. Photographs were obtained using cellSens Dimension.

Transfected cells were resuspended in serum-free DMEM medium (100 μL, containing 5,000 cells) and seeded into the upper chamber of the 24-well plate Transwell chamber (Corning, Cambridge, MA, USA), while the lower was added with 200 μl DMEM containing 10% FBS. Cells were cultured in 5% CO₂ at 37°C for 48 h, and then, the chamber was taken out, cells on the upper surfaces of the chamber were wiped off with cotton swabs, and cells from the bottom surfaces were fixed and stained with crystal violet for 15 min. Migrated cells were counted under an inverted microscope. The cell invasion assay was also conducted as described above except that the membrane was precoated with diluted Matrigel (BD Biosciences, San Jose, CA, USA). In addition, the cell seeding number for invasion experiments was 20,000 cells/well. Each experiment was conducted for three independent times.

The 24-well-plate transwell chamber (Corning Incorporated, Corning, NY, USA) pre-coated with Matrigel was for invasion assay, while the transwell chamber without Matrigel was for migration assay. Cells were cultured in the upper chamber and were allowed to transfer to the lower chamber with complete medium. Invaded and migrated cells were fixed in methanol, dyed with crystal violet, and counted under a microscope (Leica, Wetzlar, Germany).