Biocoat invasion chamber

1 × 10⁵ ESCC cells were resuspended in 300 µL of RPMI-1640 without FBS after the direct co-culture and transferred to transwell inserts with an 8-µm pore filter (BD Falcon) for migration assays and the inserts of a Corning^® BioCoat^® Invasion Chamber (Corning, Tewlbury, MA, USA) for invasion assays (Upper chambers); 800 µL of RPMI-1640 with 0.5% FBS (for assays of TE-9 or TE-10) or 0.1% FBS (for assays of TE-11) were placed in 24-well plates (Lower chambers). For assays of TE-9 or TE-10, 0.5 µM of MMP9 inhibitor (ab142180, Abcam) dissolved in dimethyl sulfoxide (DMSO) was added to the lower chambers. For assays of TE-11, 0.1 µM of MMP9 inhibitor was added to the lower chambers. Upper chambers were placed onto lower chambers and the cells were cultured for 48 h. Subsequently, the cells were fixed and stained using Diff-Quik^® (Sysmex, Kobe, Japan). Cells on the upper surface of the inserts were removed thoroughly using cotton swabs and cells that migrated or invaded the lower surface were observed under a microscope. We randomly captured five and four images in migration and invasion assays per insert at 200× magnification, respectively, and counted the number of cells. Relative migration or invasion was calculated by dividing the number of cells by that in the control (monocultured and without MMP9 inhibitor).

Cell invasion was assessed using Corning Biocoat™ Invasion Chamber (Corning, Lowell, MA, USA) following the manufacturer’s protocol. PC cells suspended in serum-free DMEM were seeded into the top well of the chamber (1 × 10⁵ cells/well) and DMEM containing 10% FBS (500 µL/well) was added into the lower chamber. After 24 h, the invaded cells on the lower side of the membrane were fixed in 95% methanol, stained with crystal violet, and photographed under an inverted microscope.

MDA-MB-231-GFP and MDA-MB-231-Omomyc cells were plated under the same conditions as the migration assay. The only difference was the use of Corning BioCoat invasion chambers, in which the porous membrane is coated with a layer of Matrigel to mimic an ECM. Cells were allowed to invade through the coated membrane for 24 hours and they were fixed, stained, and counted as explained for the migration assay. A control plate under the same conditions was also plated, and the differences in invasive capacity of the cells were corrected for the difference in cell number.