Computational protocol: LPP3 mediates self-generation of chemotactic LPA gradients by melanoma cells

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Protocol publication

[…] Cells were plated onto fibronectin-coated glass coverslips at a density of 7×104 cells/ml and then serum-starved overnight in serum-free RPMI medium prior to use as previously described (; ). Serum-free (SF) medium consisted of RPMI with 2 mM l-glutamine, penicillin/streptomycin (Invitrogen) and 0.5% BSA (Sigma), while serum-containing medium contained an additional 10% FBS. LPA-containing medium consisted of SF medium with LPA at the indicated final concentrations.Insall chambers were imaged on a Nikon TE2000-E inverted time lapse microscope equipped with MetaMorph (Molecular Devices), a motorized stage (Prior Scientific, Cambridge, UK) and Perfect Focus System enclosed in a plexiglass box humidified and heated to 37°C with 5% CO2. Each viewing bridge on the Insall chamber was imaged with a 10× objective every 30 min over 24 h.Cells were manually tracked using the MTrackJ plugin for ImageJ (http://rsb.info.nih.gov/ij). All cells on the bridge at the beginning of the experiment were tracked, excluding cells that died, to the end of the experiment or until the cells moved off the viewing bridge. Chemotactic index was calculated as described (). […]

Pipeline specifications

Software tools MetaMorph, MTrackJ, ImageJ
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Homo sapiens
Diseases Melanoma, Neoplasms, Tooth Migration