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DQ-BSA assays for proteolytic activity in lysosomes
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Cells were seeded onto 24-well plates and then treated with or without Balfinomycin A (100 nM, Sigma) for 24 h. The cells were pulse-labeled with DQ-BSA conjugated with Alexa Fluor 488 (20 µg/mL, Thermo Fisher Scientific) for 30 min. The DQ-BSA pulse labeling was followed by PBS and the corresponding chase period (0, 1, 2, 4 h). Cells were then washed again with PBS, trypsinized, and fixed with 4% PFA for 10 min. Cells were kept in cold PBS prior to FACS analysis (BD FACSCalibur). The FACS data were analyzed using FlowJo software.
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