Transwell migration assay

After co-culture for 2 days with 3T3-L1 pre-adipocutes or adipocytes, MDA-MB-231 cells were subjected to migration assay. Approximately 5 × 10⁴ MDA-MB-231 cells were suspended in 0.4 ml of serum-free medium from the co-culture system. Then, the cell suspension was added to the upper chamber, and the medium from co-culture system supplemented with 10% FBS was added to the lower chamber as a chemoattractant. Twenty-four hours later, the migrated cells were washed twice with PBS, fixed in 4% paraformaldehyde and stained with crystal violet for 30 min. The migrated cells were counted under a light microscope at ×100 magnification. Means were obtained from five randomly selected fields in each well. The experiment was repeated three times.