Radiation-Induced DNA Damage Response

Cells were seeded the day before experiments on Lab-Teks^TM (Dutscher) at a density of 3.10⁵ cells per slide and incubated at 37 °C under normoxia or hypoxia before irradiation. After radiation exposure (30 min to 24 h), cells were fixed for 15 min in 4% PFA, permeabilized, and labeled according to recommendations of the manufacturer for each primary antibody, i.e., 1 h at room temperature (1:1000, mouse anti-phospho-histone H2AX-Ser139 (Merck, Kenilworth, NJ, USA); 1:750 rabbit anti-Rad51 (Abcam, Cambridge, GB); 1:250, rabbit anti-53BP1 (NovusBio, Littleton, CO, USA); 1:250, rabbit anti-phospho-ATM-S1981 (Abcam); 1:50 mouse anti-HIF-1α (BD Transduction, San-José, CA, USA), and 1:500, rabbit anti-phospho-DNA-PKcs-S2056 (Abcam)) and secondary antibodies, i.e., for 1 h in the dark at room temperature (1:500, anti-IgG rabbit Alexa Fluor 488 (Invitrogen, Carlsbad, CA, USA); 1:250, anti-IgG mouse Alexa Fluor 555 (Abcam)). Nuclei were stained for 15 min with 1 µg/mL DAPI (Sigma, Kanagawa, Japan), then slides were mounted using Fluoromount (Merck) and stored at room temperature in the dark until analysis [38 (link)] (n = 2).