Cell transwell assays for migration and invasion

The migration and invasion capabilities of HCFBs were examined by transwell assays. A total of 1×10⁵ transfected cells/well were seeded in the transwell top chamber of an insert with serum-free media for the migration assay. The transwell top chamber was coated with 25 mg Matrigel, and then 1×10⁵ cells/well were added to the chamber for the invasion assay. The cells on the top of the membrane were removed with cotton swabs after 24 h. The invaded or migrated cells on the bottom chamber were fixed by 4% paraformaldehyde for 30 min and stained by 0.1% crystal violet for an additional 15 min. The numbers of invaded and migrated HCFBs were calculated in six randomly chosen microscopic fields (200×) per membrane. The following formula was used: relative migration/invasion cells (%) = the number of cells in experimental group / control group × 100%. All assays were performed independently in triplicate.