Computational protocol: Membrane-Lipid Therapy in Operation: The HSP Co-Inducer BGP-15 Activates Stress Signal Transduction Pathways by Remodeling Plasma Membrane Rafts

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Protocol publication

[…] B16-F10 melanoma cells were labeled with fPEG-Chol (kindly provided by T. Kobayashi) and images were taken by CytoScout fluorescent microscope. 200 nM fPEG-cholesterol (kindly provided by T. Kobayashi) labeling solution was prepared in PBS. B16-F10 cells were grown in glass-bottom dishes with two different initial cell densities: low initial cell number, LCN: 0.75×106 and high initial cell number, HCN: 6×106. In the case of BGP-15 pretreatment 10 µM BGP-15 was added to cells before the labeling for 20 min. Samples were labeled with the labeling solution for 15 min at room temperature. After washing fresh RPMI without phenol-red was added to cells and images were taken by CytoScout fluorescent microscope. Samples were illuminated in objective-based total internal reflection (TIR) configuration using 488 nm light for excitation, images were analyzed in CellProfiler (www.CellProfiler.org) and ImageJ software (http://rsbweb.nih.gov/ij/). Both the mean fluorescent intensity and the size (theoretical diameter) of each single detected microdomain were calculated. The domains we found were sorted into classes according to theoretical diameter. Diameters were calculated as 2X * (Npix/π)-2, where X is the size of a pixel in nm and Npix is the number of pixels covering the actual domain. […]

Pipeline specifications

Software tools CellProfiler, ImageJ
Application Microscopic phenotype analysis
Organisms Mus musculus, Cricetulus griseus, Homo sapiens
Diseases Melanoma, Ovarian Neoplasms, Heat Stress Disorders
Chemicals Cholesterol, Glycosylphosphatidylinositols