Boyden Chamber Assay for U87 and A172 Cell Migration

The migration of U87 and A172 cells was assayed using 24-well collagen-coated Boyden chambers (Chemicon; EMD Millipore, Billerica, MA, USA) with 8 µm pores (12 (link)). A total of 4×10⁴ cells from indicated groups (NC and PARK2 or shControl and shPARK2) were seeded in the upper chamber (0.2 ml DMEM in the upper chamber) and 0.8 ml DMEM with 10% FBS was added in the lower chamber. Following an incubation period of 48 h at 37 °C, migrating cells were quantified according to the manufacturer's protocol. Briefly, the cells that migrated to the basal side of the membrane were fixed with 4% paraformaldehyde for 5 min at 25°C and then stained with 1% crystal violet for 10 min at 25°C. The cells were subsequently visualized and photographed with a CKX41 light microscope (Olympus Corporation, Tokyo, Japan) at ×200 magnification. Images of three random fields from three replicate wells were obtained and the number of migratory or invasive cells was counted.