24 well chamber

Cell migration and invasion was investigated using the Transwell assay, and 24-well chambers (Corning Inc.). For migration, transfected HCC827 and H522 cells (10,000 cells/well) in serum-free RPIM medium were placed into the top of chambers without Matrigel™ (Corning Inc.). For invasion assays, chambers were pre-coated with Matrigel™ at 4°C overnight, and then the transfected cells (40,000 cell/well) were seeded into the upper chambers coated with Matrigel™. In either assay, RPMI medium containing 10% FBS was added to the bottom chambers. After a 24-h incubation, the migrated or invaded cells on the bottom surface were fixed with 90% methanol for 20 min and stained with crystal violet (Beyotime Institute of Biotechnology) for 10 min at room temperature. Finally, three randomly selected fields were chosen to evaluate the number of cells under a light microscope (magnification, ×100; Olympus Corporation).

For the wound healing assay, cells were planted at a density of 5×10⁶ cells/ml in a 6-well plate and incubated at 37°C overnight. 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 viewed and photographed at 0 and 24 h after scratching. Cell migration rate was calculated as follows: (original gap distance -current gap distance)/original gap distance ×100%. Transwell migration and invasion assays were examined using 24-well chambers (8 µm Transwell filters per chamber) (Corning Inc., Corning, MA, USA). Then, 3×10⁴ cells in 200 µl serum-free medium were added to the upper chamber containing an uncoated or Matrigel-coated (BD Biosciences, San Jose, CA, USA) membrane. The lower chamber contained 600 µl culture medium supplemented with 20% FBS. After being cultured for 24 h in an incubator, cells on the upper surface of the microporous membrane were wiped off with a cotton swab, fixed with 4% paraformaldehyde for 20 min, and stained with 0.1% crystal violet for 30 min. Migrated or invaded cells were counted in five randomly chosen fields in each chamber. Imaging and counting were performed at ×200 magnification under a light microscope. The experiments were executed in triplicate.

Cell invasion assays were performed in 24‐well chambers (Corning, Christiansburg, VA, USA) coated with matrigel and serum‐free RPMI 1640 medium. Cells (3–4 × 10⁴ cells/well) in 180 μL serum‐free RPMI 1640 were seeded into the upper chamber, and 600 μL RPMI 1640 with 10% fetal bovine serum was added into the lower chamber. Cells in the upper chamber were removed using a cotton swab after culturing at 37°C in a humidified 5% CO₂ atmosphere for 48 hours. Cells attached to the bottom of the membranes were fixed with paraformaldehyde and stained with 1% crystal violet. Each experiment was repeated three times.