Computational protocol: p120-catenin binding masks an endocytic signal conserved in classical cadherins

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Protocol publication

[…] Migration of cells expressing wild-type or mutant cadherins was measured using scratch wound assays. Cells were grown to confluent monolayers, scratched with a pipette tip, and imaged over time with a bright-field microscope (DM IL; Leica) equipped with a 5×/0.12 NA objective and a camera (DFC420 C; Leica). Images were acquired using FireCam software (version 3.4; Leica). For VEGF-induced migration, endothelial cells were serum starved for 1 h before being scratched, and 100 ng/ml VEGF165 peptide (PeproTech) was added 12 h after wounding. Cell replication rates were measured by incorporation of a thymidine analogue, EdU (Click-iT EdU Imaging kit; Life Technologies). For the single-cell migration assay, cells were imaged at 30-min intervals with an inverted microscope (DMI 6000B) equipped with a 10×/0.30 NA objective, a camera (Retiga EXi; QImaging), and a temperature-regulated enclosure maintained at 37°C. Phase-contrast images were acquired using Simple PCI software (version 6.6). Cell tracking data were extracted using the TrackMate plugin (version 1.2; created by N. Perry, J.-Y. Tinevez, and J. Schindelin) for ImageJ (versions 1.3–1.4; National Institutes of Health). […]

Pipeline specifications

Software tools TrackMate, ImageJ
Application Microscopic phenotype analysis
Diseases Corneal Neovascularization