Matrigel coated transwell filters

Cell motility was evaluated by the wound-healing assay; the edges of the initial scratch are indicated with a black line in the figure and the wound closure values refer to this initial position. The cell free area (percentage of control at 0 h) was then measured by ImageJ software (v. 1.8.0, National Institutes of Health Image). All experiments were performed at least three times in triplicates. Invasion was evaluated by the Transwell assay. In all, 1 × 10⁵ HCT116- and HT29-clones were resuspended in serum free cell culture media and seeded onto Matrigel-coated Transwell filters (8-µm pore size) (Costar, Corning Inc., Corning, NY, USA) coated with 200 μl of Corning® Matrigel® matrix (final concentration of 250 μg/mL) according to manufacturer’s protocol. The outer chamber was filled with 600 μl of medium containing 20% FBS and incubated at 37 °C for 48 h. Non-invading cells on the upper surface of the insert were removed with cotton swab, while those on the lower surface (invasive cells) were fixed and stained with 300 nM DAPI solution. The number of invading cells from twenty fields of each of three separate experiments was counted under the fluorescent microscope using a 10X objective and fields pictures analysed by using ImageJ software.

iPSC-RPE cells (4 × 10⁴ cells/well) were grown on Matrigel-coated transwell filters (Costar®) for one month. Paraformaldehyde-fixed cells were blocked for one hour in 10% normal donkey serum and then incubated in primary antibody overnight. The reaction was visualized using appropriate secondary antibody. Cells were cover slipped with mounting medium containing 4’,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories; Burlingame, CA, USA) and imaged with an inverted confocal microscope (Olympus FluoView FV1000).

An invasion assay was performed using an 8 µm pore size membrane coated with Matrigel (Corning Life Sciences). The MGC-803 cells (2×10⁴) in 200 µl serum-free 1640 media were seeded onto Matrigel-coated Transwell filters (Corning Life Sciences) in the upper chambers. The lower chambers contained 500 µl RPMI-1640 medium (Invitrogen Life Technologies) with 10% FBS to serve as a chemoattractant, and the chambers were incubated at 37°C in a humidified 5% CO₂. After 24 h, the non-invading cells on the upper membrane surface were removed using cotton swabs. The cells on the lower side of the membrane (invading cells) were fixed in 4% paraformaldehyde for 30 min and then stained with methylrosanilinium chloride (Sangon Biotech Co., Ltd.). The invaded cells were viewed under the Olympus BX-40 microscope and counted in five randomly selected fields (magnification, x200). The invasion abilities of the cancer cells were expressed as the mean number of cells in the five fields. The assay was performed in three independent experiments.