Fluorescence Imaging of Lysosomal and Autophagic Markers

For acridine orange (AO) stain in the screen, HeLa cells were grown in 96-well plates. Cells were treated with DMSO or indatraline, bafilomycin A1, and 658-natural chemicals for 24 h, and stained with 5 μg/mL AO. Fluorescence intensity was measured by victor plate reader, where fluorescence intensity of each well in the plate is promptly displayed as numerical readout. For confocal microscopy, 3T3-L1 or HeLa cells were grown on 15 mm coverslips at a density of 1.0 × 10⁵ cells/well in 6-well plates The cells were then treated with drugs for the time periods indicated, followed by treatment with 5 μg/mL AO (Sigma-Aldrich). Nuclei were stained with Hoechst. Following incubation for 20 min, the cells were fixed with 4% PFA and washed three times with PBS. Images were obtained using an LSM880 confocal microscope at ×400 magnification. Red fluorescence intensity was quantified using Image J2 software. For BODIPY FL-Pepstatin A stain, HeLa cells were grown on 15 mm coverslips at a density of 1.0 × 10⁵ cells/well in 6-well plates. The cells were then treated with drugs for the time periods indicated, followed by treatment with 1 μM BODIPY FL-Pepstatin A (Invitrogen) for 30 min. Nuclei were stained with Hoechst. Following incubation for 20 min, the cells were fixed with 4% PFA and washed three times with PBS. Images were obtained using an LSM880 confocal microscope at 400× magnification. For DQ-BSA analysis, HeLa cells were grown on 15 mm coverslips at a density of 1.0 × 10⁵ cells/well in 6-well plates. The cells were then treated with 10 μg/mL DQ-BSA for 2 h. After change medium, cells were treated with drugs for the time periods indicated. Nuclei were stained with Hoechst with incubation for 20 min, the cells were fixed with 4% PFA and washed three times with PBS. Images were obtained using an LSM880 confocal microscope at ×400 magnification.