Matrigel pre coating

The migratory and invasive abilities were evaluated using 8-μm Transwell inserts without and with Matrigel precoating (BD Biosciences, San Jose, CA, USA), respectively. In total, 5 × 10⁴ cells resuspended in 200 μL of DMEM were plated into the upper compartments of the Transwell inserts, and 500 μL of DMEM containing 20% of fetal bovine serum was added into the lower compartments as a chemoattractant. Following 24-h incubation, the cells remaining on the upper side of the polycarbonate membrane were gently removed with a cotton swab. Cells that moved to the bottom of the membrane were fixed with 70% ethanol, stained with 0.1% crystal violet, and rinsed thrice with double-distilled water. Finally, the migratory and invading cells were photographed and counted in at least five randomly selected visual fields under an Olympus microscope (Olympus Corporation, Tokyo, Japan), and the average was calculated.

Diluted Matrigel was used to pre-coat the Transwell chamber overnight at 4°C. Cell density was adjusted to 2×10⁵/mL in serum-free medium. We added 500 μL of medium containing 10% FBS into the basolateral chamber of the 24-well plate. We seeded 200 μL of cell suspension into the apical chamber, and 24 h later, cells were fixed in methanol for 30 min and stained with 0.1% crystal violet for another 30 min. Under an inverted microscope, invasive cells were photographed. Migration assay was conducted following the same procedures except for Matrigel pre-coating (BD Biosciences, Franklin Lakes, NJ, USA).

Transwell assay was conducted using a transwell 24-well plate (Corning, United States) with or without matrigel precoating (BD Bioscience, United States). Cells were suspended in medium without serum and added into the upper chamber. The bottom chamber was filled with 500 µL complete medium. After 48 h, the migration or invasive cells were fixed, stained with crystal violet, and then calculated.