Quantifying Oxidative DNA Damage

The method described previously [23 (link)] was followed with some modifications. In brief, cells were treated with 1 mM H₂O₂ for 1 h and harvested at the indicated time points for genomic DNA extraction. Each DNA sample was denatured at 95°C for 5 min and was then chilled on ice followed by incubation with 2 units of alkaline phosphatase (BioLabs, Ipswich, MA, USA) and 5 units of DNase I (Sigma-Aldrich) in 50 mM Tris, pH 7.3, and 1 mM MgCl₂ (Merck, Germany) buffer at 37°C for 2 h. 96-well plates were first coated with 0.003% protamine sulfate (Sigma-Aldrich) and then with 100 ng 8-OHdG (Sigma-Aldrich). Coated wells were added in a series of concentrations of pure 8-OHdG or DNA samples. The antibody to 8-OHdG (1 : 500; Trevigen), biotin goat anti-mouse IgG (1 : 1000; Zymed Laboratories, Inc., San Francisco, CA, USA), and peroxidase-streptavidin (1 : 10000; Sigma-Aldrich) were used sequentially for the detection of 8-OHdG. O-Phenylenediamine (Pierce, Thermo Scientific, Rockford, IL, USA) dissolved in citrate phosphate buffer (5.103 g citrate acid monohydrate, 7.297 g Na₂PO₄ in 1 L ddH₂O adjusted to pH 5.0 with citric acid) was used as a substrate for peroxidase. The absorbance was read at 492 nm with a microplate reader (Molecular Devices, Sunnyvale, CA, USA).