Dq red bsa dye

Lysosomal proteolytic capacity was measured using the DQ Red BSA Dye (Molecular Probes, D-12051) following manufacturer’s protocol. Briefly, 100 ul of 1mg/ml dye was added to 10 ml of warm DMEM medium. Previously plated cells in a transparent 96 well-plate were loaded with 100 ul per well each of the dye containing medium and incubated at 37°C for 1 hr. Cells were then washed twice with warm PBS and the medium was replaced with 100 µL/well of warm EBSS medium. The kinetics of DQ Red BSA digestion were recorded at respective excitation and emission maxima of 590 nm and 620 nm in a multi-plate reader over a 4 hr period.

To investigate lysosome morphology, we utilized the Lysotracker Deep Red dye (Molecular Probes, L12492) following the manufacturer’s instructions, as in ref. ^{38 (link)}. To study the proteolytic activity of lysosomes, the fluorescent DQ-Red-BSA dye (Molecular Probes, D12051) was used following manufacturer’s instructions, as in ref. ^{38 (link)}. To investigate autophagic vesicles we used the CYTO-ID® Autophagy Detection Kit in live cells, which uses a novel dye that selectively labels accumulated autophagic vacuoles. The assay was carried out following manufacturer’s instructions. Briefly, 2 μl of CYTO-ID® Green Detection Reagent were diluted in the assay buffer and then incubated for 30 min in the incubator. Imaging was carried out in complete media. In Lysotracker and Cyto-ID experiments, the number of vesicles per cell was obtained by using Cell Profiler software to assess the number of fluorescent puncta and divide it by the number of nuclei (representing the number of cells) in the same image field.