DNA Extraction and Analysis of EB-Induced Adducts

Wild type and NEIL^−/− MEF cells were grown in Dulbecco’s modified Eagle’s media (DMEM) supplemented with 10% fetal bovine serum. Cells were cultured in a humidified atmosphere of 5% carbon dioxide, 95% air, at 37 °C in 15 cm dishes until at least 70% confluency. Adhered HT1080 cells (1 × 10⁷) were treated with 500 μM EB (in triplicate) for 24 h at 37 °C. Following treatment, EB containing media was discarded, and the cells were washed twice with ice-cold phosphate-buffered saline (PBS) to remove EB. Cells were harvested by trypsin treatment, and DNA was extracted as reported previously using Qiagen Cell Lysis solution.^{16 (link)} DNA concentrations were determined by dG analysis as described below. Extracted DNA (50 μg) was subjected to enzymatic digestion and SPE enrichment for EB-FAPy-dG and EB-Gua II analysis as described above.