Validation of CellProfiler for many cellular phenotypes. (a) Cell count: for a set of 6 images of wild-type human HT29 cells (left), two researchers' counts varied by 11%, and CellProfiler's counts were within 6% of their average. For images of Drosophila Kc167 cells with various genes knocked down by RNAi (right), the two researchers' counts varied by 16% and CellProfiler's counts were within 17% of their average. Example images and CellProfiler outlines for these cell types are shown in Figure 1e. (b) Cell size: CellProfiler's cell area measurements are comparable to those of a Coulter particle counter for Drosophila Kc167 cells, for wild-type (no dsRNA) and RNA-interference induced samples. The SEM is too small to show error bars. (c) DNA content in cell populations: measurements are shown for human HT29 cell populations (1 image for each RNAi condition, left) and for Drosophila Kc167 cell populations (1,750 images for each RNAi condition were combined, right). The cell cycle distributions are as expected, with the 2N peak being predominant in the wild-type human sample, whereas most wild-type Drosophila nuclei are known to have 4N DNA content . RNAi-targeted samples were also as expected for Aurora kinase B (polyploid), Mad2 (fairly normal cell cycle distribution), String (4N-enriched), and Anillin and Cyclin A (both polyploid). (d) Chromatin content in time lapse movies: GFP-histone H4 (S. cerevisiae) or GFP-histone H2B (HeLa and C. elegans) content is shown near each nucleus in arbitrary intensity units. The histone content is decreased by roughly half in each daughter nucleus after division. For C. elegans, only the boxed region of interest was analyzed. Scale bars: C. elegans, unknown; human HeLa = 20 μm; S. cerevisiae = 10 μm. (e) Phospho-protein content: human HT29 cells treated with RNAi reagent against Polo kinase have an increased percentage of nuclei with high phospho-H3 staining compared to wild-type cells, consistent with a mitosis-stalled phenotype (left). Wild-type human HT29 nuclei that stain positively for phospho-histone H3 tend to be smaller than phospho-H3-negative cells (right). (f) Protein localization: the mean intensity of NFκB staining in the cytoplasm and the nucleus is shown in response to TNFα in human MCF7 cells (top). Totals do not equal 100% due to slight overlap between compartments. (g) Speckles: fluorescent foci of phospho-Histone2AX induced by 2 Gy of irradiation in human U2OS cells disappear at timepoints as the cells recover. Scale bar = 10 μm. The SEM is too small to show error bars.