Quantifying DNA Double-Strand Breaks by γ-H2AX

DNA double-strand breaks (DSBs) were used to evaluate DNA damage by detecting phospho-histone 2AX (γ-H2AX), a marker for DSBs [18 (link)]. PK-15 cells in 6-well plates were collected at various time points and lysed with Radioimmunoprecipitation assay (RIPA) buffer (10 mM Tris-Cl (pH 8.0), 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl, and 1 mM PMSF) and centrifuged at 12,000× g at 4 ℃ for 30 min. The γ-H2AX in the supernatant sample was detected by Western blotting using mouse anti-γ-H2AX antibody (Millipore) and alkaline phosphatase-conjugated goat anti-mouse IgG developed with NBT/BCIP (Bio-Rad).