Acridine orange staining assay

Acridine orange (AO) staining was used to visualize the volume of the cellular acidic compartments. AO is an acidotropic flourescent dye that stains DNA and cytoplasm components a bright green. Cells were treated with AO (1 μg/ml) in serum-free medium for 15 min at 37°C.

For AO staining analysis using flow cytometry, cells were washed and then illuminated with blue excitation light (488 nm). The mean fluorescence emission of green light (510–530 nm) and red light (650 nm) of 1 × 10⁴ cells were measured with a BD FACSCanto II Flow Cytometer (BD Biosciences, USA).

For AO staining analysis using fluorescence microscopy, an inverted fluorescence microscope was used (Leica DMI3000, Solms, Germany).