Measuring Cell Sensitivity to DNA Damage

To measure sensitivity, cells were treated with olaparib (JS Research Chemicals Trading, Germany), camptothecin (Topogen, Inc, US) and several chain terminators such as ABC (Carbosynth, UK), Ara-C (Sigma, USA), AZT (Sigma, USA) and ddC (Sigma, USA). Cell sensitivity to these DNA-damaging agents and chain terminators was evaluated by counting colony formation in methylcellulose plates as described previously (17 (link)). In a liquid-culture cell survival assay, DT40 and TK6 cells were treated with DNA-damaging agents in 1 ml of medium using 24-well plates and incubated at 37°C for 72 h (DT40) or 96 h (TK6). We transferred 100 μl of medium containing cells to 96-well plates and measured the amount of ATP using cellTiter-Glo (Promega), according to the manufacturer's instructions. Relative cellular sensitivity to Ara-C, ABC, AZT and ddC was measured with methylcellulose colony formation. Briefly, to evaluate the relative cellular sensitivity of each mutant to wild-type cells, sensitivity curves were drawn by setting the survival of untreated cells as 100%. The concentration of 50% viability (inhibition concentration 50%; IC₅₀) was determined from the sensitivity curves. The values of the mutant and wild-type cell lines were converted to a logarithmic scale (base 2). Each value was plotted on a bar graph.