Cls3422 48ea

Cell migration was assayed using 24-well Transwell^TM chambers with 6.5 mm-diameter polycarbonate filters with 8 μm-pore-size Transwell^TM migration inserts according to the manufacturer’s instructions with the following modifications (Corning CLS3422-48EA, Glendale, AZ, USA). The assay was based on chemotactic directional migration. Cells were treated under experimental conditions for 48 h, as were the control cells. Then, the cells were trypsinized, washed twice with serum-free medium and counted. The same numbers of living cells were seeded (2.5−3 × 10⁴ cells/well depending on the cell line) on the upper chamber and allowed to migrate through the filter to the lower chamber containing DMEM with 10% FBS. After 8–18 h (for U251MG and U87MG cells, respectively), the cells were fixed with 4% PFA for 15 min and stained with 0.5% crystal violet (Sigma-Aldrich) for 7–10 min. Cells that did not migrate through the filter were removed from the upper chamber by using a cotton swab. Migrated cells were photographed using a Nikon Eclipse Ti-U fluorescent microscope equipped with a 20× objective and DS-Qi2 digital camera and then counted with the Fiji distribution of the ImageJ 1.52a software (National Institutes of Health and the University of Wisconsin). The percentage of migrated cells was calculated by assuming 100% for the control conditions.

The migration assay was performed using 6.5 mm Transwell^® with 8μm pore polycarbonate membrane insert (Corning, CLS3422-48EA). Add 0.5 ml cell suspension (2.5×10⁴ cells) in serum-free culture medium into the upper chamber and 750μl culture medium containing 10 % FBS into the lower chambers. After 24 h, removing the cells on the upper surface of the membrane by scrubbing with cotton-tipped swabs quickly, and fixing the cells on the lower surface with pure methanol for 30 min followed by staining with analytical standard crystal violet (Sigma Aldrich, 32909) for 30min. Cell counting is facilitated by photographing the membrane through the microscope. For the invasion assay, 0.5ml cell suspension (2.5×10⁴ cells) in serum-free culture medium was added in the BioCoat™ Matrigel™ Invasion Chamber (BD, 354480), and the following procedures were in concordance with the migration assay.

6 × 10⁴ WM115 cells were placed in the upper chamber of 8 μm Transwells (Corning, CLS3422-48EA) in serum-depleted media, while the lower chamber was filled with FBS-supplemented culture media (20%). 24 h later the cells on top of the membrane were cleansed with a cotton swab while the cells that migrated on the other side were stained with Hoechst 33342 and counted.