Biocoat matrigel invasion chamber kit

For the Transwell migration assay, untransfected and transfected SGC-7901 cells in the exponential growth phase were trypsinized with 1X trypsin, washed twice with phosphate-buffered saline, and suspended in DMEM without FBS. Cells (2×10⁴ cells/well) were seeded into polycarbonate membrane inserts (8-µm pore size) in 24-well Transwell cell culture dishes. Cells were permitted to attach to the membrane for 30 min. The lower chamber was filled with 600 µl DMEM with 10% FBS. Cells were permitted to migrate for 24 h at 37°C. Following incubation, stationary cells were removed from the upper surface of the membranes. The cells that had migrated to the lower surface were fixed with 4% paraformaldehyde at room temperature for 15 min and were stained with 0.1% crystal violet for 15 min at the same temperature. The cells that had migrated through the membrane were manually counted at ×200 magnification from 5 fields/filter using a light microscope (TS100; Nikon Corporation). The invasion assay was performed using Matrigel-coated chambers from the BioCoat Matrigel Invasion Chamber kit (BD Biosciences, Franklin Lakes, NJ, USA) using the same method as aforementioned for the migration assay.

The invasive potential of the cells was evaluated using the BioCoat Matrigel Invasion Chamber kit (BD Biosciences). Briefly, the OSC-4 cells at a density of 7.5×10⁴ cells/500 µl were added to the Transwell insert chamber containing a filter coated with Matrigel. In the lower compartment, 750 µl DMEM containing 10% FBS was used as the chemoattractant. The OSC-4 cells were incubated with increasing concentrations of THP-1- or PHM-derived exosomes for 24 h at 37°C. The inserts were removed and non-invading cancer cells remaining on the upper side of the filter were scraped off. Cells that invaded the lower side of the filter were then stained with the Diff-Quick solution (Sysmex Corporation) at room temperature for 10 mins and light microscopically observed and counted in five randomly selected fields, at ×200 magnification. Each experiment was performed in triplicate.

After 24 h transfection, complete growth medium was replaced by serum-free medium. The cell migration assay was performed in 24-well transwell plates with 8.0-µm pore size membrane inserts (Corning, New York, NY, USA) whereas the in vitro invasion assay was performed using the BD BioCoat Matrigel Invasion Chamber kit (Becton-Dickinson, Franklin Lakes, NJ, USA) as previously described.9 (link)

The invasion assay was performed using a BD BioCoat Matrigel invasion chamber kit (Becton-Dickinson, Franklin Lakes, NJ, USA). TE-11 cells were separated using cell dissociation solution (Sigma) and 5×10⁴ cells/600 μl were added to the top chamber of a cell culture insert in a 24-well companion plate. After 48-h incubation, the cells that had invaded the lower surface of the membrane were fixed with methanol and subjected to Giemsa staining. The number of the migrated cells was quantified by counting them in ten random distinct fields using a light microscope.
For wound-healing assay, TE-11 cells were seeded in a 4-well chamber slide glass, and an artificial ‘wound’ was carefully created by scratching the confluent cell monolayer with the tip of a P-200 pipette. Medium with the scratched out cells were changed and then the scrambled siRNA and podoplanin siRNA were transfected into the cells. Microphotographs were taken after 0, 24 and 48 h.