Immunofluorescence for γ-H2Ax

H2Ax is a core histone protein found in the nucleosome, and the phosphorylation of H2Ax is a marker of DNA damage caused by ROS. Variant histone H2AX (γ-H2AX) is a marker of DNA double-strand breaks (Rothkamm et al., 2015). Exposed cells (1 week with 10 µM of compound) were plated in a 12-well cell culture treated plate at 1x10⁵ cells/well. Following 24 h incubation at 37°C in media containing 10 µM of compound, cells were fixed with a cold 1:1 solution of methanol and acetone for 20 min at -20°C. The cells were subsequently washed with PBS and blocked for 2 h at room temperature with 1 ml of BlockAid™ Blocking Solution (Thermo Scientific). Each well was stained with a 1:1,000 dilution of rabbit anti-phospho-H2AFX (Millipore Sigma, St. Louis, MO) in PBS overnight at 4°C with gently rocking. Following multiple washing steps with PBS, cells were incubated with 5 µg/ml of anti-rabbit Alexa Fluor 647-conjugated antibody (ThermoFisher) for 1 h at room temperature. Cells were subsequently washed with PBS and stained with 1 µg/ml of Hoechst 33342 (ThermoFisher) for 5 min at room temperature. Fluorescent images were acquired on an Evos FL Imaging System (ThermoFisher).