2.3. Long Term Survival and Colony Formation Assay

Cells were seeded into 6-well plates (5 × 10² cells/well). After 24 h of culture, the cells were exposed to radiation with doses of 2 Gy for 1 day, 2 Gy for 5 days, and 10 Gy for 1 day. After treatment, cells were cultured at 37 °C for 7–21 days. The cells were washed twice with PBS, fixed in 4% paraformaldehyde for 30 min, and stained with 0.1% crystal violet for 20 min at 25 °C. The colonies were carefully washed with tap water, and then the number of colonies, defined as >50 cells/colony, was counted and analyzed. Then the cells were washed with PBS and DMSO was added to induce a complete dissolution of the crystal violet. Absorbance was recorded at 570 nm by a 96-well-plate ELISA reader. Results were expressed as average colony count ± SE from three independent experiments.