Clonogenic Assay for Radiosensitivity

To determine the radiosensitivity of cancer cell clonogens with or without CXCR4 treatment, we seeded appropriate concentrations of single-cell suspensions in T25 flasks with the intent to generate about 50–100 cell colonies/flask. Irradiation of cells (up to 6 flasks simultaneously) was performed 6 h later when cells were attached to the flask bottom; if used, AMD3100 was added to the flasks 1 h prior to RT or 10 min after RT. Colonies of 50 cells or more developed by surviving PCa clonogens (without changing the culture medium) were fixed, stained with crystal violet, and counted 7days later. To evaluate the treatment’s effects on cell growth, we measured the relative intensity of cell staining per flask using ImageJ software.