Matrigel-based invasion assay

To reveal the effects of compound B on the invasive behavior of MDA-MB-231 cells in vitro, changes in invasiveness of cancer cells were assessed. To measure cell invasion, migration of cancer cells from a porous membrane was tested in a Transwell 24-well polycarbonate permeable support with a 8 µm pore diameter (SPL Life Sciences, Korea). The porous membrane in each well was coated with 45 µl of matrigel, an ECM gel, with a final concentration of 1 mg/ml in serum-free DMEM on ice.

MDA-MB-231 cells were treated with compound B (0.4 µM equivalent to 2 × IC_50–72h concentration) for 48 h and serum-starved overnight [15]. Following cell re-suspension, they were seeded into the upper wells (inserts) of the invasion chamber with serum free medium at a concentration of 1 × 10⁵ cells/well. Following removal of the remaining cells in the inserts after 24 h by a cotton swab, cells on the underside of the membrane were washed with PBS. Cells were then fixed using 4% formaldehyde, stained with 0.5 mg/ml crystal violet and observed under an inverted microscope.