Matrigel Invasion Assay for HepG2 and Hep3B Cells

Invasion assay was performed according to our published protocol with modification [75 (link)]. Specifically, 24-well Plate invasion chambers were pre-coated with Matrigel (Corning, Corning, NY, USA). HepG2 or Hep3B cells (5 × 10⁴), after receiving indicated treatment for 2 days, were re-suspended in 0.5 ml of serum-free medium and loaded to the upper chamber while the lower chamber was filled with 0.5 ml of complete medium containing FBS, which served as a chemo-attractant. After 16 hr of incubation at 37°C, cell ability to penetrate the extracellular matrix (ECM) was assessed by staining the cells on the lower surface of the membrane with CellTracker™ Green (Molecular Probes, Eugene, OR, USA). Four fields of cells were counted randomly in each well under a fluorescent microscope at 200× magnification. Data are normalized to control treatment, which is set as 1, and are expressed as mean ± SEM of three independent experiments.