Invasion Transwell and clonogenic assays

After glioma cells grew to 70–90% confluence, cells were serum-starved for 24 h and then diluted at a cell concentration of 1×10⁵ cells/ml. Cell suspension (500 µl; 5×10⁴ cells) was added into the upper Transwell chamber, and 600 µl 10% FBS-DMEM was pipetted into the lower Transwell chamber. Transwell plates were then cultured in the Matrigel-coated inserts in a humidified incubator (37°C, 5% CO₂) for 48 h. Non-migrated cells on the upper side of the membrane were removed using a cotton swab. The cells were then fixed with 1 ml methanol at room temperature for 20 min and stained with 0.1% crystal violet for 10 min at room temperature. In total, six fields per Transwell were imaged using an inverted microscope at ×10 magnification, and the migrated cells on the lower site of the membrane were counted. Each condition in the experiment must be performed at least in duplicate.

For the clonogenic assay, after glioma cells grew to 70–90% confluence, cells were serum-starved for 24 h and then diluted at a cell concentration of 1×10⁵ cells/ml. Cells were seeded into 6-well plates at a density of 500 cells/well and then cultured for 5 days, followed by crystal violet staining for 20 min at room temperature. The cells were washed twice with PBS and the blue colonies were counted using a light Nikon ECLIPSE E100 microscope at a magnification of ×10. The data were expressed as % survival relative to the control. Each condition in the experiment was performed at least in duplicate.