Acridine orange staining

Acridine orange (AO), a lysosomotropic weak base that accumulates in intracellular acidic vesicles due to proton trapping, is usually used to measure the functional state of lysosomes.²⁰ It is also a concentration dependent meta-chromatic fluorescent dye. Upon the excitation by blue light, AO can be visualized as red fluorescence at high concentrations (in intact lysosomes) and green fluorescence at low concentrations (in the cytosol and the nucleus). Thus, AO relocation, from lysosomes to cytosol, and the decrease of granular (lysosomal) red fluorescence (dimer form) in combination with the increased diffuse (cytosolic) green fluorescence (monomer form) may imply the deterioration of lysosomal membrane stability with a decreased proton gradient, which permits the leakage of the lysosomal contents to cytosol.²⁰ After incubated with 0.5 μM Pb (12 h) or 50 μM CQ (3 h) or EBSS medium (2 h), respectively, cells grown on coverslips in 24-well plates were loaded with 5 μg/ml AO at 37 °C for 30 min, rinsed twice with warm (37 °C) PBS and examined under confocal laser scanning microscope (TCS SPE, Leica, Germany) with excitation at 488 nm. Green fluorescence (emission peak between 530 and 550 nm) and red fluorescence (emission peak at about 650 nm) were simultaneously collected by two separate windows.