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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 (, 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