Colony formation assay

To determine clonogenic survival after irradiation, the glioblastoma cell lines U-373 MG and U-87 MG were treated with photon doses of 0 to 8 Gy. One day before irradiation, 150 cells in 3 ml medium were disseminated per well of standard 6-well tissue culture plates and incubated at 37°C and 5% CO₂. After irradiation, the cells were incubated under identical conditions until most of the colonies in the control sample (non-irradiated cells) had reached a size of more than 50 cells. Then, the cells were fixed with freezing-cold methanol for 3 minutes. The fixed cells were then stained with crystal violet for 45 minutes. Colonies >50 cells were counted under a light microscope.