Computational protocol: Loss of strumpellin in the melanocytic lineage impairs the WASH Complex but does not affect coat colour

Similar protocols

Protocol publication

[…] Fibronectin was diluted in PBS to 10 μg/ml and used to coat glass bottom 6‐well plates for 2 h at 37°C. Coated dishes were washed in PBS. Immortal melanocytes were trypsinized and seeded at 50 000 cells per well of a 6‐well plate and left to adhere overnight. Images of cells were taken every 30 min for 24 h using a Nikon TiE timelapse microscope. Individual cells were tracked using the mTrackJ plugin for ImageJ, and spider plots were generated using the Chemotaxis Tool plugin for ImageJ. [...] For WASH intensity analysis, fixed exposure images of cells immunostained with anti‐WASH were outlined using the ROI tool in ImageJ and the fluorescent intensity for the entire cell was calculated using the ‘measure’ function in ImageJ. For colocalization analysis, the subtract background function of imageJ was applied to each channel of the image individually and using only the WASH channel image a 10 × 10 μm box was placed over the strongest signal at the cell centre. The Coloc 2 plugin for ImageJ was run using this ROI, and the resulting Pearson ‘R’ value was used as a measure of WASH colocalization with various markers. […]

Pipeline specifications

Software tools MTrackJ, ImageJ, Coloc
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus
Diseases Spastic Paraplegia, Hereditary