Computational protocol: Simultaneous Visualization of Both Signaling Cascade Activity and End-Point Gene Expression in Single Cells

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Protocol publication

[…] Image J 1.44 d was used to make maximum intensity projections (MIPs) of the image stack of each channel. In the FITC channel, ImageJ was applied to enhance the contrast, subtract background (rolling ball radius = 200 pixel) and run a median filter (radius 4 pixel). Background was also subtracted from DAPI-images (rolling ball radius = 50 pixel).Subsequently, CellProfiler v.10415 was used to identify cell nuclei (thresholding method: two-class thresholding “Otsu PerObject”) in previously background subtracted DAPI images. The nuclei were then combined with background corrected FITC images to identify the cytoplasm of individual cells (method to identify objects: “Watershed - Image”, thresholding method: two-class thresholding “Otsu Adaptive”). Signals in Cy3 (PDGFRβ) and Cy5 (DUSP6 or ACTB) were enhanced (feature type: “speckles”, feature size: 10), counted (diameter: 2–20 pixel, thresholding method: “RobustBackground Global” (for Cy3), diameter: 1–20 pixel, thresholding method “RobustBackground Adaptive” (for Cy5), threshold correction factor: 0.8) and related to the individual cells. Subsequently, if images did not contain signals, the amount of signals had to be manually corrected, since then the adaptive enhancement of Cy3 and Cy5 signals gave rise to false positive signals. Graphs were done in R . In images were for better visualization treated in ImageJ as described here: One slice of the DAPI z-stack was selected, background was subtracted in ImageJ and thresholded for brightness and contrast. Cy3 and Cy5 z-stacks were projected to MIPs, the background was removed, brightness and contrast adjusted and a maximum filter (size: 1 pixel) was applied. […]

Pipeline specifications

Software tools ImageJ, CellProfiler
Application Microscopic phenotype analysis
Organisms Homo sapiens