Alkaline comet assay.

The alkaline comet assay, examining DNA repair activities in gel, was performed as described previously (29). Cells were trypsinized and diluted to ∼1 × 10⁵ cells/ml, and 250-μl aliquots of the cell suspension were placed into the wells of a 24-well plate on ice. The cells were treated with hydrogen peroxide (12.5 μM) in suspension for 5 min and then embedded in 1% low-melting-point agarose (Bio-Rad, Hemel Hempstead, United Kingdom) on a microscope slide precoated with 1% normal-melting-point agarose and allowed to set on ice. The slides were placed in a humidified chamber to allow the cells to undergo DNA repair in gel for up to 2 h at 37°C prior to being placed in freshly prepared cell lysis buffer containing 2.5 M NaCl, 100 mM EDTA, 10 mM Tris-HCl, pH 10.5, 1% (vol/vol) dimethyl sulfoxide (DMSO), and 1% (vol/vol) Triton X-100 for at least 1 h at 4°C. The slides were transferred to an electrophoresis tank and incubated in the dark for 30 min in fresh cold electrophoresis buffer (300 mM NaOH, 1 mM EDTA, 1% [vol/vol] DMSO, pH 13) to allow the DNA to unwind. Electrophoresis was performed at 25 V and 300 mA for 25 min, and the slides were neutralized with three 5-min washes with 0.5 M Tris-HCl (pH 8.0) and allowed to air dry overnight. The slides were rehydrated for 30 min in water (pH 8.0), stained for 30 min with SYBR Gold (Life Technologies, Paisley, United Kingdom) diluted 1:20,000 in water (pH 8.0), and allowed to air dry prior to imaging. The cells (50 per slide; 2 slides per time point) were analyzed using Komet 6.0 image analysis software (Andor Technology, Belfast, Northern Ireland). Percent tail DNA values were averaged from the results of at least three independent experiments.