Matrigel coated invasion chamber

5 × 10⁴ CRC cells were suspended in serum-free DMEM and seeded on the upper compartment of Matrigel-coated invasion chambers (Corning) with an 8-μm pore polycarbonate membrane. The lower compartment was filled with 10% FCS-DMEM. After 24-h incubation, cells on the upper side of the membrane were removed using a cell scraper. Migrated cells on the lower side of the membrane were fixed with 95% ethanol, stained with 0.1% crystal violet solution, and counted with a light microscope.

To investigate the cell invasion ability, the transwell assay was performed with 24‐well Matrigel‐coated invasion chambers (Corning). Briefly, the stable monoclonal cell lines (2.0 × 10⁵ cells per well) were resuspended in the medium without FBS and placed in the upper compartment of transwell chambers with fibronectin coated on the lower surface. The lower compartment was filled with 700 μL medium containing 10% FBS as a chemoattractant. Cells were fixed in 4% formaldehyde and stained with 0.1% crystal violet after 24 h. Ten fields were randomly selected and counted under a light microscope (Carl Zeiss).

The invasive potential of pancreatic cancer cells was assessed in vitro in matrigel-coated invasion chambers (Corning; NY, USA) in accordance with the manufacturer's instructions. Briefly, cells in the log growth phase were serum starved for 24 h prior to seeding, detached by brief trypsinization and resuspended in medium containing the appropriate treatment. The matrigel invasion inserts were rehydrated and prepared as described in the manufacturer's instructions. Cells in serum-free medium at a density of 1 × 10⁵ cells/ml/well were placed on the top of matrigel-coated polycarbonate filters (8 μm pore size) suspended in a membrane invasion culture system chamber; the chamber underneath the membrane contained complete medium. The cells were incubated in a CO2 incubator at 37^°C for 5 h, after which the non-invasive cells were removed from the upper surface of the membrane and the invasive cells on the undersurface of the membrane were fixed and stained with hematoxylin–eosin (H&E; Sigma-Aldrich). These experiments were performed in triplicate at least three times. The invading cells were counted under a fluorescence microscope in five random high-power fields.

For migration assays, 24-well transwell chambers with 8.0 µm PET membranes were used and for invasion assays, Matrigel-coated invasion chambers with the same specification were used (Corning). A375 cells were serum-starved overnight before plating 2.5 × 10⁴ cells per chamber in 200 μL serum-free DMEM medium. Wells were filled with 700 μL DMEM containing 10% FCS to encourage migration. Cells were incubated for 18 h for migration and 24 h for invasion assays. Following incubation, migrated cells attached to the reverse side of the membrane were fixed with methanol for 10 min and stained with Giemsa stain solution (Sigma). Three images were captured per technical replicate (in triplicate) using a light microscope with a camera (Leica Application Suite), and cells were counted manually using ImageJ (with three independent biological replicates of each cell line carried out).

For the cell-invasion assay, 1 × 10⁵ cells were added to the upper well of Matrigel-coated invasion chambers (8-μm pore size; Corning Inc., Corning, NY, USA) for 24 h. Next, the cells on the upper side of the membrane were wiped off, and the cells on the lower side of the membrane were fixed in cold methanol and were stained with 0.2% crystal violet (Sigma, St. Louis, MO, USA). The invasive cells were counted under 5 high-power fields in each chamber. An artificial wound was introduced into monolayer cells using a sterile 200-μl pipette tip. The wound closure was monitored and photographed at 0 and 24 hours. The migration ability was evaluated as the distance of wound closure in 5 high-power fields.

Cells were seeded in 8 μM matrigel coated invasion chambers (Corning, Corning, NY) with NMI or clorgyline, for 22 hours. Subsequently invaded cells were stained using the hemacolor kit (EMD Millipore, Massachusetts) and imaged. Stained cells were counted manually for analysis.