Computational protocol: Quantum dots implementation as a label for analysis of early stages of EGF receptor endocytosis: a comparative study on cultured cells

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Protocol publication

[…] All data were obtained from at least three independent experiments. In each experiment, 4–5 fields containing 15–20 cells totally were imaged for each time point. The images were processed and analyzed using Leica Confocal Software (Germany) and ImageJ software (National Institute of Health). In confocal images, the background of each channel was subtracted and, in some cases, brightness/contrast was adjusted only for presentation. No filter was applied in quantitative analyses. Z-stacks were projected onto single images (projections) using the max intensity method (ImageJ). Then, single sections or projections were exported to Adobe Photoshop 5.0 for final image processing.Analysis of Region of Interest (ROI) intensities was carried out using ImageJ. The number and integrated intensities of endosomes were measured from max intensity projections using ImageJ (menu command Analyze). The quantitative co-localization analysis was performed using ImageJ JACoP Plugin [] to determine Manders' coefficient (M1: red pixels overlapping green pixels). Thresholds were set by a visually estimated value for each channel.For all quantitative analyses, the results are presented as the mean ± 95% confidence interval for at least fifteen cells. The column charts were created using Microsoft Office Excel 2007. […]

Pipeline specifications

Software tools ImageJ, JACoP
Application Microscopic phenotype analysis
Diseases Neoplasms, Myoclonic Epilepsies, Progressive
Chemicals Biotin, Sodium