4.4. Matrigel Invasion Assay

Analysis of the Matrigel invasion chamber (pore size: 8 mm, BD Bioscience, Lowell, MA, USA) was performed according to the manufacturer’s protocol. After inoculating the cells in a 6-well plate at a density of 1 × 10⁶, they were treated with Bazedoxifene with 1, 5, or 10 μM for 24 h. After 24 h, 5 × 10⁵ cells were inoculated into the upper chamber with serum-free medium, and complete medium (10% FBS, 1% penicillin–streptomycin) was added to the lower chamber. Matrigel chambers were incubated for 48 h in an incubator set at 37 °C and 5% CO₂. After 48 h, non-invasive cells on the top of the chamber were removed with a cotton swab. In order to determine the number of invading cells under the filter, the cells were stained using a dyeing reagent (Diff Quik, Kobe, Japan) and the stained cells were counted using ImageJ software.