Matrigel coated transwell chamber

Cell invasion assay was performed using Matrigel-coated transwell chambers (8 μm pore size; Costar). A total of 100 μL transfected cell suspension (2 × 10⁴ cells/mL) was added to the upper chamber of the transwell plates. A total of 500 μL complete medium was added to the bottom chambers. After incubation for 24 h at 37 °C, nonmigrating cells were removed using cotton swabs. The cells that permeated the Matrigel-coated membrane and migrated to the bottom were fixed using paraformaldehyde and then stained using crystal violet.

To assay cell invasiveness, Matrigel-coated transwell chambers (Corning Costar) were used. Equal numbers of cells maintained in pH 7.4 or pH 6.5 were suspended in RPMI of each pH condition containing 1% FBS. Approximately 200 μL of the cell suspension was added to the upper portion of the insert, and medium containing 5% FBS was added to the lower portion of the inset. After 8 h (for AGS) and 18 h (for SNU601) of incubation at 37 °C in 5% CO₂, noninvasive cells were removed from the upper surface of the transwell membrane with a cotton swab, and the invaded cells on the lower layer surface were fixed in 4% formaldehyde and stained with crystal violet solution. The numbers of invaded cells were counted or imaged under high-power (×200) microscope magnification (Olympus). For the migration assay, cells incubated at pH 7.4 and pH 6.5 were sampled in the same manner as above and grown in 24-well plates in growth medium. After overnight culture, the middle of the cell surface was scraped with a micropipette tip to make a wound of constant width. Debris was washed out with PBS, and the wound closures were monitored and photographed at 6 h (AGS cells) and 18 h (SNU601 cells) under the microscope (× 100, Olympus). Migration distance was calculated applying the software program HMIAS-2000.

Cell invasion was evaluated by Matrigel-coated Transwell chambers (Corning-Costar, NY, USA). Briefly, cells were incubated in the upper chamber of a 24-well Transwell plate. After transfection for 48 h, 5 × 10⁴ cells were seeded into the upper compartment, which contained RPMI1640 medium with 10% FBS. After 24 h incubation, non-invading cells were gently removed and the migrated cells were fixed with methanol followed by DAPI staining. The number of invaded cells was counted under light microscope (Nikon, ECLIPSE Ts2, Japan).

Cell invasion was analyzed with Matrigel-coated transwell chambers (Costar, Corning, NY, USA). Cell migration was tested using transwell chambers without Matrigel and wound healing assay. For transwell assay, CAL27 and SCC25 cells (1 × 10⁴ for migration assay, 3 × 10⁴ for invasion analysis) in serum-free DMEM were plated into the superior chambers, whereas the lower chamber was supplemented with 500 μL DMEM with 10% serum. 24 h upon incubation, migrated or invasive cells were spotted with 0.1% crystal violet, and examined under a microscope (× 100 magnification, Nikon, Tokyo, Japan) with at least 6 random fields.
For wound healing assay, 2 × 10⁵ CAL27 and SCC25 cells after various transfections were placed into each well of 6-well plates until 80% of confluency was reached. Next, a “wound” was made using a 200 μL pipette tip, and cells were allowed to migrate for additional 24 h. The images (× 100 magnification) at 0 and 24 h were recorded. Relative migratory rate was calculated by the formula: (si-circ_0011946_{(wound area)0h})-(si-circ_0011946_{(wound area)24h})/(si-NC_{(wound area)0h})-(si-NC_{(wound area)24h}).

For transwell invasion assays, Caski and Caski/Taxol cells were plated atop Matrigel-coated transwell chambers with 8 μm pores (Costar Corp.) in serum-free media after being cultured in serum-free RPMI-1640 medium for 48 h. In the bottom chamber, media supplemented with 10% FBS was added. A total of 72 h after cell plating, the transwell chamber was extracted and fixed in 4% formaldehyde in room temperature for 20 min, washed with PBS twice, and put into well with 400 μl Giemsa A solution for 1 min in room temperature; 800 μl Giemsa solution was added into the well for 5 min; the transwell chamber was extracted and washed with PBS twice. The cells in the upper chamber were cleared using clean cotton tips, the lower chamber was dried and moved to slides, and three fields were observed under microscope, and the average number of cells was calculated. Each treatment was assessed in triplicate.