Two methods of cell viability were employed : cell-counting using an automated cell counter (NanoEnTek, Seoul, Korea) after trypan blue staining and the clonogenic assay. During the survival assay, cells were plated on 60-mm culture plates (2×104 cells). After 24 hours, 10 μM dexamethasone was added, and 24 hours later, the cells were subjected to different doses of radiation : 0, 2, 5, 10, 15, and 20 Gy. Cells were then incubated at 37°C with regular media changes for 8–10 days until the control was 100% confluent. Cells were washed twice with phosphate-buffered saline (PBS), trypsinized with 1 mL trypsin-EDTA, stained with trypan blue, and counted using an automated cell counter. The percentage of viable cells was calculated from the ratio of treated cells to normal control cells. Three independent experiments were carried out, and the mean value was determined.
For the colony-forming assay, cells were plated at clonogenic density (1.0×103) in a 60-mm culture plate, then treated with dexamethasone (10 μM) and a radiation dose (10 Gy) as per the experiment setup. Cells were incubated for 8–10 days with regular media changes after treatment. Cells were fixed with 4% paraformaldehyde for 20 minutes, then stained with 0.4% crystal violet for 20 minutes, washed under running distilled water, and allowed to dry for 2 hours. A cluster of 50 cells or more was scored as a colony.
For the colony-forming assay, cells were plated at clonogenic density (1.0×103) in a 60-mm culture plate, then treated with dexamethasone (10 μM) and a radiation dose (10 Gy) as per the experiment setup. Cells were incubated for 8–10 days with regular media changes after treatment. Cells were fixed with 4% paraformaldehyde for 20 minutes, then stained with 0.4% crystal violet for 20 minutes, washed under running distilled water, and allowed to dry for 2 hours. A cluster of 50 cells or more was scored as a colony.