Clonogenic Survival Assay after UV-C Exposure

Cells were plated at single cell density in 6 cm dishes with 275 cells per dish and clonogenic cell survival was assayed similarly to as described previously (5 (link),22–24 (link)). Cells were allowed to adhere overnight following standard culture conditions, washed with 1× DPBS (Thermo Fisher Scientific #14190-144), and exposed to indicated dose of UV-C (254 nm) radiation before the culture medium was replenished and cells were returned to the temperature and CO₂-controlled incubator. Fourteen days after exposure, cells were fixed with 4% paraformaldehyde (Fisher Scientific #50-980-487) diluted in 1× PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.4) for 15 min at room temperature. The fixative was removed and cells were washed with 1× PBS and then stained with 0.5% crystal violet (Fisher Scientific #C581-25) diluted in water for 60 min. Excess stain was removed and dishes were washed with water. Dishes were imaged using a E-Gel Imager (Thermo Fisher Scientific). Colonies (defined as a minimum of 50 cells) were counted using FIJI. Percent survival was calculated as the percentage of colonies that grew on the treated dishes relative to the untreated dishes at each indicated dose.