DQ Red BSA assay for visualization of lysosomal degradation

Lysosomal proteolytic degradation was evaluated using DQ Red BSA (#D-12051; Thermo Fisher Scientific), a fluorogenic substrate for lysosomal proteases, that generates fluorescence only when enzymatically cleaved in intracellular lysosomal compartments. hiPSC-derived neurons were seeded at a density of 400,000 cells/well of a Matrigel coated 48-well plate. After 24 h, cells were washed once with DPBS, treated with complete media containing either 10 µg/ml DQ Red BSA or vehicle (PBS) and incubated for 6 h or 24 h [16, 72, 90] at 37 °C in a 5% CO₂ incubator as described in [54]. At the end of 6 or 24 h, cells were washed with PBS, fixed with 4% PFA and immunocytochemistry was performed as described in “Methods”. Cells were imaged using a Leica SP8 confocal microscope and all image processing was completed with ImageJ software. Cell bodies were identified by MAP2 labeling, and fluorescence intensity of DQ Red BSA was measured in regions of the images containing the MAP2 label.