Figure 3

Glioma cell motility. U251 glioma cells were irradiated (2 Gy) and incubated in serum-free medium for 72 h, irradiated conditioned medium (IR-CM) then was collected from the supernatant of culture dishes and used for cell motility assays. Cell motility was evaluated using 24-well chambers with an 8-μm pore polyethylene teraphthalate (PET) membrane coated with matrigel for invasion assays, and non-coated PET membranes for migration assays. The lower compartment contained 0.75 ml of serum-free medium (control), IR-CM and serum-free medium with VEGF (5 ng/ml) (A). To neutralize VEGF activity in IR-CM, IR-CM was supplemented with monoclonal anti-human VEGF antibody (1, 10 and 20 μg/ml) (B). 5 × 10⁴ cells were placed in top chamber, incubated for 22 h and glioma cells transversing the membrane were fixed and stained. Stained cells were counted by light microscopy in nine randomly chosen high-power fields (x 200). Images were captured by a Photometrics Sensys CCD camera and imported into IP Labs image analysis software package. Columns, mean from three independent experiments; bars, ± SD.