Transwell filter chamber

Transwell filter chambers (Corning Inc., NY, USA) were used to analyze the differences in vertical migration and invasion of cells in each group. For the migration assay, cells were trypsinized and resuspended in 200 μL of DMEM at the upper level of a transwell chamber (2 × 10⁴ cells/well), and 600 μL complete medium (DMEM: FBS = 4:1) was added to the bottom level. After 24 h, cells on the outer surface were fixed with 4% paraformaldehyde for 30 min, and non-migrated cells on the inner surface were removed using a cotton swab. Finally, cells were stained with crystal violet for 15 min and counted by selecting 5 random fields of view using an inverted microscope. For the invasion assay, 100 μL of Matrigel (BD Biosciences) was pre-coated evenly on the membrane of the upper chamber, otherwise, the experiment was the same as the migration assay.

MDCK II, HEK293, and L cells, all of which were kindly provided by Dr. M. Takeichi, were maintained in Dulbecco’s modified Eagle’s medium (DMEM) or in a 1:1 mixture of DMEM and Ham’s F-12 medium supplemented with 8% fetal calf serum (FCS) and antibiotics. Polarized MDCK II cell cultures were established by plating the cells at 2 × 10⁶ cells in 100-mm Transwell filter chambers (Corning), and the cells were incubated for 7 days with a daily change of fresh culture medium. The integrity of the monolayer was verified by measuring the Trans-epithelial Electrical Resistance with a volt-ohm meter (Millipore). MDCK II and HEK293 cells stably expressing wild-type mouse Wnt3a, mutant Wnt3a (Wnt3a (S209A)) or wild-type mouse Wnt11 were established by transfection with wild-type Wnt3a, Wnt11 or Wnt3a (S209A) plasmid as indicated previously50 (link). In these cells, Wnt genes were expressed under the control of the CMV promoter. Cells were selected and maintained in culture medium containing 400 μg/mL and 200 μg/mL G418, respectively. L cells stably expressing wild-type Wnt3a were established as previously described41 (link). The supernatant from cultures of Wnt3a- or Wnt11-producing cells was prepared as previously described50 (link).

Transwell invasion assays using 8 μm polycarbonate Transwell filter chambers (#353097; Corning, Corning, NY, USA) in 24-well plates were performed as previously described [30 (link)], with minor modifications. The top surfaces of the Transwell membranes were coated with Matrigel^® Matrix. The cells were seeded at 50,000 cells·well⁻¹ into the top chambers of the Transwell plates. To minimize the effect of cell proliferation, the cells were fixed12 h after being seeded in the upper chamber.

Cell migration and invasion ability were assessed through Transwell filter chambers (Corning, New York, NY, USA), which were 8-mm pore size chamber inserts in a 24-well plate. For the migration assay, 48 h after the IC30 or IC50 ebastine treatment, 200 μL of serum-free medium containing 5×10⁴ MNNG and U2OS cells or 1×10⁵ MG63 cells was dropped into the upper chambers. For the invasion assay, 1×10⁵ MNNG, U2OS cells or 2×10⁵ MG63 cells were placed into the upper chambers, which were coated with Matrigel diluted in serum-free culture medium. Separately, 800 μL of culture medium supplemented with 10% FBS (fetal bovine serum) was added to the lower chambers. The control group was treated with an equal amount of DMSO. After incubation at 37°C, the cells on the bottom surface of the membrane were stained and counted.

The in vitro endothelial tube formation assay was performed using Geltrex^™ reduced growth factor basement membrane matrix (Invitrogen). Briefly, HUVECs suspended in CM were plated on a culture plate coated with Geltrex^™ matrix and incubated at 37°C in a 5% CO₂ atmosphere overnight. Each culture was photographed at x 100 magnification using a camera connected to an inverted microscope. The WIMtube image analysis platform (WIMASIS GmbH, Munich, Germany) was used to quantify total tube length. Matrigel invasion assays were performed using transwell filter chambers (8-μM pores, Corning Inc., NY, USA). HUVECs suspended in EGM^®-2 MV Single Quotes^® media were inoculated into upper transwell chambers coated with Matrigel (BD Bioscience). The lower chamber was filled with prepared CM. Invaded HUVECs on the bottom surfaces of the upper chambers were stained with Hemacolor^® Rapid staining solution (Merck Millipore), and cells remaining in the upper chambers were removed with a cotton swab. Stained cells were observed under a light microscope and counted in 5 selected fields.

For cell migration assay, cells were harvested and re-suspended in DMEM with 1% FBS. Then, 2 × 10⁴ cells were seeded into the upper chamber of transwell filter chambers (Corning, 4.5-μm pores), and the lower chambers were filled with 2 mL DMEM containing 10% FBS. After 18 hours of incubation, cells that migrated into the reverse side of the transwell membrane were fixed with methanol, stained with Crystal Violet, and then counted under an inverted light microscope.

The migration or invasion assays were performed using polycarbonate Transwell filter chambers (8 μm pore size; Corning Inc.) and the inserts were coated with or without Matrigel® (BD Biosciences). A total of 2 × 10⁴ cells were seeded in the upper transwell chamber and 1 × 10⁵ cells were added in the top chamber containing Matrigel with 100μL serum-free medium, whereas the bottom chamber was added with 500μL medium containing 10% serum. After being cultured for 48 h, the migrated cells on the lower membrane were stained with 0.1% crystal violet (KGA229, Keygen Biotech) and counted.