Computational protocol: Differential Rac1 signalling by guanine nucleotide exchange factors implicates FLII in regulating Rac1-driven cell migration

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Protocol publication

[…] Stromal collagen fibre organization and crosslinking in fibroblast-collagen matrices was assessed using GLCM analysis. This method allows the study of the texture of a sample and provides a readout of the level of crosslinked collagen organization, as previously described. GLCM analysis was performed in ImageJ software as outlined in . [...] Cells grown on coverslips in the presence of ethanol (−dox) or 1 μg ml−1 dox (+dox) for 24 h were fixed in 4% formaldehyde for 15 min and incubated for 3 min with cell permeabilization buffer (0.5% Triton-X (v/v) in PBS) at room temperature. Cells were next blocked in 1% bovine serum albumin (BSA; Roche, 10735078001) in PBS for 30 min before incubation with relevant primary antibodies (in 1% BSA) for 1 hour at room temperature. The respective fluorophore-conjugated secondary antibodies were then added for an additional hour at room temperature in the dark. Antibodies used for immunofluorescence are outlined in . Coverslips were mounted onto slides using the ProLong Gold antifade reagent with DAPI stain (Invitogen, P36935) and images were captured using the low light microscope system and processed with Metamorph software (Molecular Devices). Images shown were analysed in ImageJ. Additional details on the specifications of microscopes used for image recording are described in . […]

Pipeline specifications

Software tools ImageJ, MetaMorph
Application Microscopic phenotype analysis
Diseases Neoplasms