Lysosomal V-ATPase activity was assessed with acridine orange staining. acridine orange uptake represents lysosomal V-ATPase-driven pumping of hydrogen ions into the lysosomes36 (link). After the indicated treatments, HEI-OC1 cells were washed with PBS and incubated with 1 μM acridine orange (ThermoFisher, A1301) in the cultured medium for 30 min at 33 °C. The acridine orange was then removed, and the cells were examined under a confocal microscope (LSM 880, Zeiss, Germany). The cytoplasm and nuclei of stained cells showed bright green fluorescence, whereas the acidic autophagic vacuoles showed bright red fluorescence10 (link),37 (link).
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