Transwell chambers with 8 m pore size polycarbonate membrane

24-well Transwell chambers with 8 µm pore size polycarbonate membrane were used (Corning Inc., Corning, NY, USA) for cell migration and invasion assays. For migration assay, 1.0×10⁵ cells were planted on the top side of the membrane. For invasion assay, 1.0×10⁵ cells were planted on the top side of the membrane precoated with Matrigel (BD Bioscience). After 24 hrs, the cells that had passed through the membrane to the lower surface were fixed with methanol, stained with crystal violet, and then counted.

Transwell chambers with 8 µm pore size polycarbonate membrane (Corning, NY, USA) were inserted into a 24-well plate. For the migration assay, 2×10⁴ cells were resuspended in 100 μL of serum-free medium and filled into the upper chambers. Meanwhile, 600 μl of RPMI1640 complete medium was supplied into the bottom chambers. Then, cells were incubated at 37°C with 5% CO₂ for 24 hrs. For the invasion assay, 100 μL of Matrigel was pre-coated on the upper chamber. Following steps of the invasion, assay were the same as the abovementioned. Cells were fixed in 4% phosphate-buffered paraformaldehyde and stained by 0.5% crystal violet solution (Beyotime, Nantong, China) after the incubation. At last, the numbers of migrated and invasive cells were pictured by calculating from five random fields per sample.

Cell migration and invasion assays were performed using 24-well Transwell chambers with 8-µm pore size polycarbonate membrane (Corning, Cambridge, MA, USA). The transfected cells were cultured for 48 h and then 5×10⁴ cells were seeded on the top side of the membrane without Matrigel for the cell migration assay or pre-coated with Matrigel (BD Biosciences) for the cell invasion assay. A total of 500 µl DMEM containing 20% FBS was added to the lower chambers. Following incubation in 12 h for the migration assay or 48 h for the invasion assay, cells inside the upper chamber were removed with cottons swabs. Migrated and invaded glioma cells attached to the lower membrane surface were fixed with 75% methanol for 30 min at room temperature, and then stained for 30 min with crystal violet at room temperature. Five random fields of view at ×100 magnification were counted in each well under a light microscope and expressed as the mean number of migrated or invaded cells. Three independent assays were performed.