(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). elegans, only the boxed region of interest was analyzed. The histone content is decreased by roughly half in each daughter nucleus after division. elegans) content is shown near each nucleus in arbitrary intensity units. cerevisiae) or GFP-histone H2B (HeLa and C. (d) Chromatin content in time lapse movies: GFP-histone H4 (S. 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). 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. (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). (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. Example images and CellProfiler outlines for these cell types are shown in e. 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. (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. Validation of CellProfiler for many cellular phenotypes. Cells touching the border are intentionally excluded from analysis and images were contrast stretched for display. (e) Outlines show the identification of nuclei and identification of cell edges made by CellProfiler in human HT29 (left) and Drosophila Kc167 (right) cells. The results are comparable to those produced by white referenced images (right), but they do not require the error prone and often omitted step of collecting a white reference image immediately before image acquisition. (d) These corrections reduce noise in quantitative measurements, demonstrated here in DNA content measures (middle) from images of Drosophila Kc167 cells that are improved over the raw images (left). Images were contrast-enhanced to display this effect. CellProfiler's illumination correction modules correct these anomalies (right). (c) Image processing example: uneven illumination from the left to the right within each field of view is noticeable in this three row by five column tiled image (left).
(b) Schematic of a typical CellProfiler pipeline. (a) Main CellProfiler interface, with an analysis pipeline displayed.
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