Wound-Healing and Transwell Assays

For conducting wound-healing assay, cells were plated and grown until reaching 90% confluence in six-well plate. The confluent monolayer of cells was scratched using a pipette tip. After 24 hr, the cells migrating into the wounded areas were observed. Images were captured using an inverted microscope at 0 hr and 24 hr after scratching. Then, for performing the migration of GBM cells, briefly, cells were plated on the top chamber of transwell assay inserts (Costar, Cambridge, MA, USA) with a membrane containing 8-µm pores in 200 µl serum-free medium, and the lower chamber contained medium with 10% fetal bovine serum. After 24 hr, the cells that did not migrate on the upper surface of the membrane were gently removed with a cotton swab. The cells on the lower surface were fixed with 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet for 20 min. Migrated cell from five random fields were counted under an inverted microscope using a magnification of 200×. For invasion assay, the steps were similar to those of migration assay, except that the Matrigel (BD Biosciences, San Jose, USA) was added into the membrane. The assays were conducted in triplicate.