Rs 2000 biological system irradiator

Exponentially growing rectal cancer cells cultured in 100 mm dish were irradiated at 0.61 Gy/min per exposure by X-ray irradiator (RS 2000 Biological System irradiator, Rad Source, USA). After irradiation, the cells were harvested for clonogenic survival analysis. Survival after radiation exposure was defined as the ability of the cells to maintain their clonogenic capacity and to form colonies. Briefly, cells were trypsinized, counted, and seeded 500 cells per well into 6-well plate. After incubation for 15 days, colonies were stained with 0.5% crystal violet solution for 30 minutes and counted manually. Experiments were performed at least 3 times independently.

Cell irradiation was performed using an X-ray irradiator (RS 2000 Biological System irradiator, 25 mA, 160 kV; Rad Source Technologies Inc.) by administering a single 6Gy dose of radiation. The cells were exposed to single irradiation of 6Gy once a week, 35*35cm irradiation field, 100cm source skin distance, 1cm compensator, and a rack angle of 180°. After radiotherapy, cell culture dishes were placed in an incubator to continue the culture until the next radiotherapy. The H460 parent cells (H460-PT) were treated with a total of 72 Gy ionizing radiation and considered as radioresistant cells (H460-RR).

Human fetal lung fibroblasts (MRC-5) and human umbilical vein endothelial cells (HUVECs) (Cell Bank, Chinese Academy of Sciences, China) were, respectively, cultured in minimal essential media (MEM) and Dulbecco's minimal essential media (DMEM) supplemented with 10% fetal bovine serum (FBS, Gibco) and were maintained in a humidified incubator with 5% CO₂ at 37°C. Cells were irradiated using a X-ray irradiator (RS 2000 Biological System irradiator, Rad Source, USA).