Transwell plate

As previously reported [22 (link)], we evaluated cell migration and invasive capabilities of HCC cells with Transwell plates (Millipore, Billerica, MA, USA).

Cell migration assay was carried out by using transwell plates (Millipore, Billerica, USA) as described previously with a little modification. MDA-MB-231 cells and MCF-7 cells were transfected with LOXL2, stimulated with TNF-α/TGF-β or hypoxia (a modified incubator chamber flushed with a gas mixture containing 1% O₂, 94% N₂ and 5% CO₂ in a humidified atmosphere). Then, they were treated with escin Ia (2.5, 5, 10 μM) or BAPN (100 μM) for 6 h, and detached and suspended in culture medium. Then, cells (1 × 10⁴ cells/well) was added into the upper chamber of the transwell plates, while the lower chamber was full of 600 μL culture medium with 10% FBS as a chemoattractant. After being incubated for 6 h at 37°C, the non-migrated cells on the upper surface of the membrane were removed by soaked cotton swab. In addition, the cells migrated to the bottom face of the membranes were counted after being stained with crystal violet solutions. Then, fields per filter were captured randomly at a magnification of 200 × with Olympus IX51 inverted microscope.

The migration and invasion abilities of PTC cells were assessed using transwell plates (Millipore, Billerica, MA, USA). PTC were seeded in uncoated (migration assays) or Matrigel-coated (invasion assays) with a diameter of 8 μm (BD Bioscience, Bedford, MA, USA). The upper chamber was seeded with cells at a density of 2 × 10⁴ cells/well in medium without serum, and FBS with 10% serum was added to the lower chamber. For invasion assays, Matrigel-coated chambers were used. After 24 h of incubation, non-migrating cells on the top surface of the filter were removed by rubbing with a cotton swab and cells that had migrated to the lower chamber were quantified in five random fields using an optical inverted microscope at a magnification of 200× (Nikon, Tokyo, Japan).

The 48-well Transwell plates (Millipore, Bedford, MA, USA) with 8 μm pore filters were used for measuring cell migration. A total of 1 × 10⁶/well PDAC cells were seeded in the upper chambers and then incubated with medium alone at 37 °C in a 5% CO₂-filled incubator. PDAC cells migrating to the lower surface were stained with 0.5% crystal violet (Sigma-Aldrich, St. Louis, MO, USA) and photographed.

To observe the ability of the supernatants of scratch-injured HUVECs on THP-1 cell migration and invasion, chemotaxis and invasion assays were performed in 8 µm transwell plates (Millipore, MA, USA).
In the chemotaxis assay, 5 × 10⁵ THP-1 cells in 100 µL volume of culture were added to the upper chamber. In the invasion assay, 40 µL of diluted Matrigel was put into the upper chamber of a 24-well transwell and incubated at 37°C for 1 hour. Then, 1 × 10⁶ THP-1 cells in 100 µL volume of culture were added to the upper chamber. Afterwards, the supernatants were added to the lower chamber of the transwell for both chemotaxis and invasion assays. After 24 hours of incubation, cells were washed with PBS and fixed with paraformaldehyde. Cells were then stained with Giemsa (Sigma, MO, USA) for 5 minutes. The results of the chemotaxis and invasion assays were evaluated by counting the number of migrating or invading cells under a microscope.