8 μm polycarbonate nucleopore filters

For the cell migration assay, the cells were seeded in the transwell unit with 8‐μm polycarbonate nucleopore filters (Corning Costar). The upper chamber contained serum‐free medium and the medium in lower compartment contained 10% FBS; the cells were kept in the incubator for 24 hours. The cells in the lower surface of the filter were fixed and calculated. For the cell invasion assay, the membrane of the transwell unit was coated with 40 μL Matrigel (BD, USA) at 37°C for 4.5 hours to form a basement membrane and the cells were treated in a similar fashion as that in the cell migration assay.

To examine the migratory ability of cells in vitro, the transwell chamber assay was performed. Cells were placed in the upper chamber of a 24-well transwell unit (5 × 10⁴ cells/well) with 8-μm polycarbonate nucleopore filters (Corning Costar, Cambridge, MA). The upper compartment contained serum-free medium while the lower compartment contained medium with 10% fetal bovine serum; the cells were incubated for 24 h in a humidified atmosphere of 5% CO₂ at 37°C. The cells adhering to the lower surface of the filter were fixed and counted. The cells from at least five representative fields were analyzed. For the invasion assay, the membrane of the transwell unit was coated with 40 μl Matrigel (BD Biosciences, San Jose, CA, USA) at 37°C for 4 h to form a reconstructed basement membrane. The cells were treated in the same way as the migration assay.
A wound healing assay was also applied to evaluate the cell migration ability. Cells were seeded in 3.5-cm plates and grown to a density of 70–80%. Then, a 200-μl pipette tip as used to create an artificial wound of scratched cells. The migrating distance was measured after 48 h.

The 8-μm polycarbonate nucleopore filters (Corning Costar, U.S.A.) were used in cell migration and invasion assays. For migration analysis, osteosarcoma cells (1 × 10⁵ cells) were seeded in the upper chamber of a 24-well Transwell unit. The upper chamber contained serum-free medium and the lower compartment contained medium with 10% FBS. After 24-h incubation, the cells adhering to the lower surface of the filter were fixed and stained with crystal violet (Beyotime, China). The staining cells were counted in five representative fields.
For the invasion assay, the Transwell membranes were precoated with Matrigel (BD Biosciences, U.S.A.) at 37°C for at least 4 h to form a reconstructed basement membrane. The procedure was the same with migration assay.

To carry out migration assays, we plated the miR-143 mimics or corresponding NC-transfected TPC-1 cells into the top 24-well Transwell chamber (5 × 10⁴ cells/well) containing the 8-μm polycarbonate nucleopore filters (Corning Costar, Corning, NY, USA). The top chamber was added with a serum-free medium, while the bottom had a 10% FBS-containing medium. Later, we incubated cells for 24 h under 37°C with 5% CO₂ conditions. We then fixed cells attached to the lower filter surface and counted them with crystal violet staining.
In invasion assays, Matrigel (40 μL, BD Biosciences, USA) was coated on the Transwell chamber ahead of time, followed by 4 h incubation under 37°C for the formation of the basement membrane. The remaining analyses were identical to those in the migration assays.