An image-based assay that identifies EDR within cells. An image-based assay that identifies EDR within cells. SW480 cells were incubated for 48 hours at 37°C and 5% CO2 in medium containing 0.4% (65 mmol/L) DMSO alone or with 3 μmol/L podophyllotoxin. After addition of Hoechst's DNA stain and incubation for 1 hour at ambient temperature, the cells were imaged by a laser scanning microtiter plate cytometer. Scans of a single well from a 1,536-well plate are shown for DMSO minus or plus podophyllotoxin-treated control wells. Fluorescent objects were classified and color-coded as follows: nuclei with ≤4N DNA content [dark blue, 3,000–50,000 fluorescence units (FLU), 40%–100% Gaussian shape)] nuclei with >4N DNA content (red, 50,000–200,000 FLU, 40%–100% Gaussian shape), unrecognizable as single nuclei (cyan, 3,000–200,000 FLU, <40% Gaussian shape), and excluded fluorescent objects (yellow, <3,000 or >200,000 FLU). Guassian shape refers to the fit of the intensity profile to an ideal sphere (100%). Histograms using these color classifications were constructed from 16 wells each treated with DMSO alone (−podophyllotoxin) or with 3 μmol/L podophyllotoxin (+podophyllotoxin) to reveal the frequency of each type of object. The results are analogous to a FACS profile in which the relative numbers of cells in G1, S, and G2/M phases of the cell cycle are determined. Wenge Zhu et al. Mol Cancer Res 2011;9:294-310 ©2011 by American Association for Cancer Research