Evaluating Lysosomal V-ATPase Activity

Lysosomal V-ATPase activity was assessed with acridine orange staining. acridine orange uptake represents lysosomal V-ATPase-driven pumping of hydrogen ions into the lysosomes^{36 (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 fluorescence^{10 (link),37 (link)}.

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Zhao T., Zheng T., Yu H., Hu B.H., Hu B., Ma P., Yang Y., Yang N., Hu J., Cao T., Chen G., Yan B., Peshoff M., Hatzoglou M., Geng R., Li B, & Zheng Q.Y. (2021). Autophagy impairment as a key feature for acetaminophen-induced ototoxicity. Cell Death & Disease, 12(1), 3.

Publication 2021

acridine orange LSM 880

Acidic Acridine orange Atpase Autophagic vacuoles Cells Confocal microscope Cytoplasm Fluorescence Lysosomal Nuclei of cells

Corresponding Organization :

Other organizations : Binzhou University, Binzhou Medical University, Women & Children's Hospital of Buffalo, Shenzhen Second People's Hospital, Nanjing Tech University, Second Affiliated Hospital of Xi'an Jiaotong University, Guangdong Pharmaceutical University, Case Western Reserve University

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Variable analysis

independent variables

Indicated treatments

dependent variables

Lysosomal V-ATPase activity (assessed with acridine orange staining)

control variables

PBS washing
Incubation time (30 min)
Incubation temperature (33 °C)
Acridine orange concentration (1 μM)

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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