Cell Adhesion Assay

A 96-well plate was coated with PBS (as negative control) and 10 μg/ml of laminin, vitronectin, and fibronectin, over 4 h at 37°C. To block any remaining protein binding sites on the plate, coating solutions were removed, and then 1% bovine serum albumin (BSA) was added for another hour. Appropriate density of GBM cell suspension (10,000 cells/well for GBM6; 5,000 cells/well for U87MG and HK308) was seeded, followed by another 2 h incubation. Non-adherent GBM cells were removed by careful washing two times with PBS. Then, 100% ethanol was used to fix the adherent cells for 15 min followed by 0.1% crystal violet (Thermo Fisher Scientific, Pittsburgh, PA, United States, dissolved in 100% ethanol) staining for another 30 min. After excessive stain was removed and 0.3% Triton-X was applied to lyse cells, the absorbance was measured at 570 nm using a microplate reader (BioTek, Winooski, VT, United States). The percentage of adhesion was determined by dividing the corrected (background subtracted) optical density of adherent cells by the total corrected optical of cells added to each microplate well and multiplying by 100%. Experiments were repeated three times with five replications per experiment.