Comparison of ELISA with other DNA damage assays.

<p><b>(A)</b> HT29 cell cultures were treated with the indicated drugs for 1 hour (with the exception of cisplatin, with which cells were treated for 6 hours). Drugs were added to cultures at the following concentrations: SN38, vorinostat, erlotinib, and romidepsin at 1 μM; LMP744 at 10 μM; and cisplatin and etoposide at 100 μM. Samples were then divided into two aliquots; one was prepared for microscopy and stained for γ-H2AX (green) and DNA (blue; DAPI) and the other was extracted and analyzed by ELISA. The % γ-H2AX values from the ELISA assay are shown in the bar graph above the stained IFA images. The dashed horizontal line indicates the value for vehicle. <b>(B)</b> Representative microscopy images from A375 xenograft mice treated with individual drugs or drug combination as indicated. A fragment of each xenograft was fixed and processed for γ-H2AX immunofluorescent staining (IFA). γ-H2AX staining is green; nuclei are stained with DAPI blue. <b>(C-E)</b> Bar graphs showing quantification of γ-H2AX IFA staining <b>(C)</b>, % γ-H2AX ELISA readings <b>(D)</b>, and PAR ELISA readings <b>(E)</b> in xenograft tumors. Mice were treated as indicated on the bottom of each figure. Readings from individual mice are shown on the left (top for <b>E</b>) and averages of the treatment cohorts (n = 6 for all) are on the right (bottom for <b>E</b>).