Flow Cytometry

Cells were seeded into 6-well plates at a density of 2 × 10⁵ cells/well. Cells either exposed to radiation (8 Gy) or not were treated with DMSO or DQ (1 mM D+20 mM Q) for 24 h, digested with trypsin, and collected. Cells were then resuspended in a 100-μl binding buffer with 1-μl fluorescein isothiocyanate Annexin-V and 1-μl propidium iodine (PI; BD Biosciences, 556547). Finally, samples were analyzed by flow cytometry (C6, BD Biosciences, San Jose, CA). For cell cycle analysis, cells were fixed with Fixation/Permeabilization Diluent/Concentrate (eBioscience) for 30 min. Subsequently, intracellular Ki-67 (eBioscience) and Hoechst33342 (Sigma) staining were performed using PermWash solution (eBioscience). Cells were washed once prior to flow cytometry analysis.