Extracellular matrix gel

Wound healing assay was used to evaluate cell migration ability. Serum was withdrawn before analysis to avoid effect of cell proliferation. The migration status was assessed by measuring the movement of cells into the scratched area created by a 10 μl pipette tube, and the spread of wound closure was observed at indicated times, then photographed under a × 10 objective lens.
Cell invasive ability was determined using transwell chambers coated with a extracellular matrix gel (BD Biosciences). 5 × 10⁴ cells/well were placed in the pre-coated upper chamber and cultured with serum-free DMEM. The lower chamber contained 300 μl complete media. After 48 h incubation, the cells on the upper surface of the filter were removed. Then the filters were fixed with 4% paraformaldehyde for 15 min and stained with crystal violet stain for 30 min (Sigma). The invasive activity was quantified by counting the number of trespassed cells per five high-power fields (magnification, × 100) from three independent experiments (mean±S.D.), and a t-test was used to show significant differences between two groups (P<0.05).

A total of 3 × 104 cells were re-suspended in 200 μl of serum-free medium and placed in the upper compartment of a Transwell chamber (Corning; 24-well insert, pore size: 8 μm) with (Invasion assay) or without (Migration assay) extracellular matrix gel (BD Biosciences, San Jose, CA, USA). The lower chamber was filled with 15% fetal bovine serum as a chemoattractant and incubated for 48 h for the migration assay. The cells on the upper surface of the membrane were removed, and the cells on the lower surface were fixed and stained with 0.05% crystal violet. Five visual fields of each insert were randomly chosen and counted under a light microscope.

We used transwell inserts (24-well inserts, 8 um pore size; Corning Inc. Corning, NY, USA) to detect the invasive capacity of cholangiocarcinoma cells in vitro. The inserts for the invasion assay were pre-coated with extracellular matrix gel (BD Biosciences, Bedford, MA, USA). The transfected TFK-1 or HUCCT-1 cells were cultured with serum-free medium overnight. The following day, transfected cells were re-suspended with medium containing 0.1% bovine serum albumin and transferred to the upper chambers of the transwells. The lower chambers were filled with medium containing 10% FBS. Then the cells were incubated at 37 °C for 48 h. Later, the cells traversed through the membrane and adhered on the lower surface. Then, 0.4% paraformaldehyde and 0.1% crystal violet were used to fix and stain the cells, respectively. Finally, the stained cells were counted under a light microscope. Experiments were performed in triplicate.

Matrigel invasion assay was carried out to assay the cells invasive capacity with a chamber containing a polycarbonate membrane (8 μm pore size) and coated with a layer of extracellular matrix gel (BD Biosciences) according to the manufacturer’s instructions. After 72 h transfection, the number of cells that penetrated the matrigel was counted from six randomly selected fields per membrane.