Matrigel Invasion Assays

The invasiveness of the MDA-MB-231 cells was evaluated using BD Matrigel Invasion Chamber (8-μm pore size; BD Biosciences, Bedfrord, MA, USA) according to manufacturer's instructions as previously described (32). MDA-MB-231 cells (1 ×10⁵) were suspended in 0.5 mL of media containing vehicle or the indicated concentrations of carnosol were seeded into the upper chambers of the system, while DMEM supplemented with 10% fetal bovine serum was placed into the bottom wells in the system as a chemo-attractant and then incubated at 37°C for 24 h. Non-penetrating cells were removed from the upper surface of the filter with a cotton swab. Cells that have penetrated through the matrigel to the lower surface of the chamber were fixed with 4% formaldehyde, stained with DAPI, and counted under a fluorescent microscope. For quantification, the assay was done in duplicates and repeated three times.