γ-H2AX foci formation assay

1 × 10⁵ cells were cultured in a flask to achieve 70% confluence. The flask was fixed with 2% paraformaldehyde followed by 0.5% Triton X-100 permeability (Sigma, MO, USA). Suspended cells were incubated with a mouse monoclonal primary antibody target to Ser 139 phosphorylation of H2AX (Cell Signaling Technology, USA, 1:400) at a room temperature for 4 hours and were incubated with Alexa Fluor 488-labelled secondary antibody (Cell signaling1:1000). Cells were finally placed on the microscope slide with a cover slip containing a mounting medium, and samples were analyzed using a fluorescence microscope (Zeiss, Germany).