8 μm transwell filters

Cell migration was examined using a BioCoat Matrigel Invasion Chambers equipped with 8-μm transwell filters (BD Bio-sciences). LADC cells (5 × 10⁴) were suspended in the upper chamber with serum-free medium, whilst medium containing 10% FBS was placed in the bottom chamber as a chemo attractant. After incubation for 24 h at 37°C all cells on the upper surface of the membrane were removed and stained with coomassie brilliant blue followed by manual counting under a light microscope in three randomly selected fields of view (Olympus, Tokyo, Japan). All procedures were performed in triplicate.

For the in vitro wound-healing assay, a cell-free area of the culture medium was wounded by scratching with a 200-μL pipette tip. Cell migration into the wound area was monitored in a serum-free medium and photographed using a fluorescence microscope at 0, 24, and 48 h. Then, the effects of fibrinogen (Sigma, St. Louis, MO) on cell migration and invasion were determined using 8-μm transwell filters (BD Biosciences, Franklin Lakes, NJ, USA) with or without Matrigel (BD). GBC-SD (3 × 10⁴) and NOZ (4 × 10⁴) cells in 0.5 μL of serum-free DMEM and William’s media were added to the upper chamber, which contained a non-coated/Matrigel-coated membrane. The same protocol was performed on the treatment group (40 μg/mL fibrinogen). The lower chamber was filled with 500 μL of basal medium comprising 10% fetal bovine serum. After a 24-h incubation at 37°C in a 5% CO₂ humidified incubator, cells that migrated to the lower compartment were fixed with methanol and stained with crystal violet. Migrated or invaded cells were counted in five randomly chosen fields in each well, and imaging and cell counting were performed at 10× magnification using a fluorescence microscope. The experiments were performed in triplicate.