Matrigel coated insert

In vitro invasion assays were performed in triplicate using an invasion assay kit with Matrigel-coated inserts (BD Bioscience, San Jose, CA, USA) as described previously.^{24 (link)} A volume of 1 × 10⁶ cells per well was added to the upper compartment of the invasion chamber with or without pharmacological inhibitors in transfected cells. To the lower compartment, we added serum-free conditioned medium with or without attractants. In vitro migration assays were performed in triplicate using a 48-well microchemotaxis chamber as described previously.^{25 (link)} Type I collagen-coated polyvinyl pyrrolidine-free polycarbonate filters with 8 μm pore membranes (Neuro Probe, Gaithersburg, MD, USA) were used in these modified Boyden chambers. After the samples were incubated for 6 (for migration) and 17 h (for invasion) at 37 °C, the migrated or invaded cells were sequentially fixed, stained with Diff-Quik reagents (Dade Behring Inc., Newark, DE, USA) and quantitated by counting the number of cells in five random high-power fields for each replicate (× 200) with a light microscope.

The ability of thyroid cancer cells to invade was measured using Transwell chambers with Matrigel-coated inserts (BD Biosciences, San Jose, CA, USA). Cells in serum-free medium were added to the upper chambers at a density of 2 × 10⁵ cells/well. RPMI-1640 (Sigma-Aldrich) containing 10% FBS was added to the lower chambers. Following incubation at 37°C for 48 h, noninvading cells were wiped off with a cotton swab, and invading cells were stained with 0.1% crystal violet and photographed under a microscope. Five fields were randomly selected to count the number of invading cells.