For tracking of lysosomes and mitochondria, cells were double-stained live with 50 nM Lysotracker Red DND-99 (Molecular Probes Cat. No. L-7528) and 50 nM Mitotracker Deep Red FM (Molecular Probes Cat. No. M22426). For measuring mitochondrial membrane potential (and tracking as well), cells were stained with 200 nM Mitotracker JC-1 (Molecular Probes Cat. No. M34152). Trackers were added directly to culture supernatants and incubated for 1 h at 37 °C. Imaging was then performed without further washing of cells. Live imaging of compartmentalized axons in Xona Microfluidic Chambers (MFC) was performed with a Leica HC PL APO 100 × 1.46 oil immersion objective on an inversed fluorescent Leica DMI6000 microscope enclosed in an incubator chamber (37 °C, 5% CO2, humid air) and fitted with a 12-bit Andor iXON 897 EMCCD camera (512×512, 16 µm pixels, 229.55 nm/pixel at 100Χ magnification). For more details, refer to https://www.biodip.de/wiki/Bioz06_-_Leica_AFLX6000_TIRF . Excitation was performed with a TIRF Laser module in epifluorescence (widefield) mode with lines at 488, 561 and 633 nm. Fast dual color movies were recorded at 3.3 frames per second (fps) per channel over 2 min (400 frames in total per channel) with 115 ms exposure time as follows: Lysotracker Red (excitation: 561 nm, emission filter TRITC 605/65 nm) and Mitotracker Deep Red (excitation: 633 nm, emission filter Cy5 720/60 nm) or for Mitotracker JC-1 with excitation at 488 nm and fast switching between emission filter FITC 527/30 nm (green channel for compromised membrane potential) and TRITC 605/65 nm (red channel for intact membrane potential). Movie acquisition was performed at strictly standardized readout positions within the micro channels of the micro groove barrier that separated the proximal seeding site from the distal axonal exit as illustrated in Fig. 2a . Specifically, the readout windows were located either just adjacent to the channel exit (distal readout) or the channel entry (proximal readout).
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