Matrigel coated membrane

The migration and invasion abilities were determined using a Transwell apparatus without or with Matrigel-coated membranes (BD Biosciences, Franklin Lakes, NJ). The migration and invasion assay procedure was detailed in a previous study [11 (link)]. The total number of attached cells was counted. All experiments were performed in triplicate. Single cell migration assay was performed as following description. Mock and stably SPANXA-expressing CL1-5 cells were seeded 5000 cells/per well in 6-well culture plate for overnight attachment. Wash wells with PBS twice and replace fresh medium. Set up the focus and sites for 25 position /per well, and take the picture every 1 hour in 24 hours. The high content screening software MetaMorph (Molecular Devices, Sunnyvale, CA) was used to analyze every single cell tracking and mean velocity.

To further confirm the role of yCsCB4 in human cancer cell growth, wound-healing assays were performed to evaluate the effect of yCsCB4 on cell migration according to the previous method [17 (link)]. MHCC-97H and RBE cells seeded in 6-well plates were grown to 80 % confluence and incubated with yCsCB4 (1 μg/ml) or PBS for 24 h. Cells were wounded by scratching with pipette tips. Wounds at 24 h were observed and photographed under a light microscope (Leica DMI3000B).
To evaluate the effect of yCsCB4 on cell invasion, we performed transwell assays according to the method described elsewhere [18 (link)]. MHCC-97H and RBE cells were suspended in serum-free media and placed in 8 μm pores. These inserts were placed in wells with serum-containing media. Cells were incubated with yCsCB4 (1 μg/ml) or PBS for 24 h. Invasion assays were performed using matrigel-coated membranes (BD, USA). The migration assay was similar to the invasion assay, except that the upper side of the membranes was not coated with the matrigel. Cells attached to the lower surface of the membranes at 24 h were counted under a light microscope.

Transwell assay inserts (8 μM PET, 24-well Millicell) or Matrigel-coated membranes (BD Biosciences) were add with 200 μL serum-free medium in the upper chamber and 800 μL serum-containing medium in the lower chamber. Totally 50,000 cells were added to the upper chamber and cells in lower chamber were fixed and stained after 24 h (migration) or 48 h (invasion).

For the wound-healing assay, 5 × 10⁵ cells were seeded into 6-well plates and grown to confluence. Mitomycin C (10 μg/ml) was used to suppress cell proliferation before scratching. Wounds were created by scraping the confluent cell monolayers with a 10-μl pipette tip. After being extensively rinsed to remove cellular debris, the cells were cultured in a serum-free medium. The wound closure rate was monitored every 12 h, and images were taken using an inverted microscope TE-2000S (Nikon, Tokyo, Japan). A Transwell invasion assay was performed in a 24-well Transwell plate with 8-μm polyethylene terephthalate membrane filters (Corning Costar Corp, Corning, NY, USA); 1 × 10⁵ cells in 200 μl of serum-free medium were added to the upper chambers, which contained Matrigel-coated membranes (BD Biosciences). Each lower chamber was filled with a 500-μl medium with 10% FBS. After 18 or 24 h of incubation, cells that invaded the bottom chamber were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Invasive cells were counted in five randomly chosen fields (magnification, ×200) per well.

Cell monolayers in individual wells were scraped with a sterile pipette tip (100 μl) after the different groups of ICC cells (1 × 10⁶ cells/well) were grown to 90% confluence. The cells were incubated for 24 h and photographed with a digital camera (Leica, Heerburg, Germany) at 0 h and 24 h after wounding. The distance of cell migration into the denuded area was used to determine the degree of wound healing [24 (link)].
The different groups of ICC cells (1 × 10⁴ cells/well) were seeded in the upper chambers of 24-well Transwell plates containing Matrigel-coated membranes (BD Biosciences, San Jose, CA, USA). Complete medium was added to the bottom chambers. After 36 h of incubation, uninvaded cells were removed from the top surface of the membrane in the upper chamber with a cotton swab. Invaded cells on the underside of the membrane in the upper chamber were fixed with 4% paraformaldehyde, stained with 1% crystal violet and counted under a microscope in a blinded manner [24 (link)].

For evaluation of cell migration, we used 24-well, 6.5 mm internal diameter transwell plates (BD Biosciences) with Matrigel-coated membranes (8 μm pore size) separating the 2 chambers. SW579 and TPC-1 cells after the appropriate transfection were seeded in non-serum media in the upper chamber at 50,000 cells per well. 10% FBS medium served as chemoattractant in the lower chamber. 24 h post-seeding, non-invading cells were removed and invaded cells were fixed with methanol. After crystal violet (0.1%) staining, images were captured by Olympus CKX41 at 100× magnification and registered using the getIT software (Olympus).