Transwell chambers 24 well insert 8 μm pore size

Cells were cultured in 6-well plates at 1 × 10⁶ cells/well in a medium without FBS. When the cells reached 70–80% confluence, they were wounded by scratching with a sterile pipette tip, washed at least three times with phosphate-buffered saline (PBS), and cultured in Opti-MEM^® (Gibco, Life Technologies, Beijing, China). At 0, 12, 24, 36, and 48 h after being wounded, the cells were observed and images were obtained using a DFC300FX microscope (Leica, Jena, Germany).
For transwell migration and invasion assays, we used non-coated or coated membranes in transwell chambers (24-well insert; 8-μm pore size; Corning Life Sciences, Corning, NY, USA). Cells (1 × 10⁴) were plated in the top chamber and cultured with no serum medium after transfection in 6-well plates for 24 h. A medium containing 10% FBS was added to the lower chamber. After incubation at 37°C in 5% CO₂ for 36 h, unpenetrated cells in the upper chamber were wiped with a cotton swab, and the penetrated cells were fixed in methyl alcohol for 20 min and subjected to hematoxylin–eosin (H&E) staining 10 and 5 min, respectively, for counting. Cell numbers were counted under a DFC300FX microscope.

In wound-healing assay, the cells were cultured in 6-well plates at 3 × 10⁵ cells/well in FBS-free medium. When cells reached 70–80% confluence, sterile pipette tips were used to scratch and form a “wound”. In transwell migration and invasion assay, we used non-coated and coated membranes in transwell chambers (24-well insert; 8-μm pore size; Corning Life Sciences, Corning, NY, USA) respectively. Preprocessed cells (3 × 10⁴) were added in the upper chamber with FBS-free medium and medium containing 10% FBS was added to the lower chamber. After cultured in cell incubator for 48 h, a cotton swab was used to wipe the unpenetrated cells in the upper chamber, and the penetrated cells were fixed in methyl alcohol for 20 min and then subjected to hematoxylin and eosin staining 10 min and 5 min respectively for counting. The “wound” and the stained cells were observed and photographed via a DFC300FX microscope (Leica, Jena, Germany). The width of “wound” and cell number were further obtained using Image J software (NIH, Bethesda, MD, USA).