In cell workstation software

Neural aggregates (~day 27–31) were dissociated using Accutase and plated onto poly-ornithine/laminin coated 96-well plates at 25,000 cells/well. 16 hours later, media was changed to L CM for 24 hours. Cells were then incubated in L CM + 10 uM EdU for 4 hours, followed by incubation in L CM (without EdU) for 4 hours. Cells were then fixed as described above, and EdU staining was carried out using a Click-iT EdU Imaging Kit (Invitrogen).
To quantify the EdU signal, cells were imaged using an IN Cell Analyzer 2000 high content platform (GE Healthcare). Image sets were analyzed using the multi-target analysis algorithm in the IN Cell Workstation software (GE Healthcare).

Neurons were plated 50,000 per well on 96-well imaging plates (Greiner) coated with poly-L-lysine (Sigma). At 10 DIV, neurons were labeled with LysoTracker Red (Invitrogen) according to the manufacturer’s specifications, and 20 ng/ml of DAPI. Labeled live cells were imaged at 40x magnification using the IN Cell Analyzer 2000 (GE Healthcare). Cell count, lysosome count, mean lysosome area and total lysosome area were calculated. Four fields per well were acquired using the DAPI and Cy3 channels for visualizing the DAPI and LysoTracker Red, respectively. Images were analyzed with the IN Cell Workstation software (GE Healthcare) multi target analysis protocol. Briefly, nuclei were segmented by applying a Top Hat algorithm with a minimum area of 50 square μm and a sensitivity level of 50 to the DAPI channel. An 8 μm collar around the nuclei was used to distinguish the perinuclear region and the distal region of the cells (Fig. 3E, F). Lysosomes were defined as objects with a 1 to 3 μm diameter segmented by 2 scales with a sensitivity level of 20 in the corresponding channel. Lysosomes were categorized as perinuclear or distal according to the region defined by the collar. Independent experiments were performed on WT and G2019S KI neurons plated as described above. Cells were fixed and subject to Lamp-2 immunocytochemical staining and high content image analysis.