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Protocol publication

[…] CAD cells (catecholaminergic neuronal tumor cell line, RRID:CVCL_0199) were maintained in DMEM/F12 medium with 10% Fetal Bovine Serum (). Cell line identity was confirmed by the morphological response to serum withdrawal (i.e. neurite formation), a previously-documented characteristic of this cell line (). 24 hr before the experiment cells were plated on coverslips in serum-free medium to induce neurite formation. Cells were treated with inhibitors or control solvent (0.1% DMSO final in all experiments) for 60 min, then lysosomes were labeled with 0.1 µM (final concentration) LysoTracker Red DND-99 (Molecular Probes) and used for microscopy immediately after LysoTracker addition.Microscopy was performed using a Nikon TE-2000 inverted microscope equipped with a LED X-Cite illuminator (Lumen Dynamics) at 37 degrees. Images were acquired with a Hamamatsu ORCA-Flash 4.0 camera driven by MetaMorph software at a rate of 1 frame per second for 1 min. Lysosome motility in neurites was analyzed using Diatrack software (http://www.diatrack.org/), particle velocities were measured from frame to frame and the total length of trajectories divided by the number of analyzed organelles was calculated for every experimental condition. […]

Pipeline specifications

Software tools MetaMorph, DiaTrack
Application Laser scanning microscopy