Lysosomal Membrane Permeability Assay

Acridine orange is a versatile fluorescence dye that easily crosses the cell membrane and reversibly accumulates into acidified membrane-bound compartments, such as lysosomes. Acridine orange gives fluorescence emission in a concentration-dependent manner, which is red at high concentrations (e.g., in lysosomes) to green at low concentrations (e.g., in the cytosol), with yellow as intermediate (e.g., upon trapping in nucleoli). The ratio of red-to-green emission in comparison with controls may thus either monitor lysosomal leakage or change in lysosomal pH. rPMs were grown in 96-well culture plates. rPMs were first exposed with acridine orange (5 μg/ml) at 37°C for 15 min, which were rinsed, then incubated in HBSS with or without cART and NAC for the indicated times. Cells were examined at 1 h intervals using a Synergy™ Mx Monochromator-Based Multi-Mode Microplate Reader (BioTek Instruments, Inc. Winooski, VT, USA) with excitation wavelength at 485 nm and emission recorded at 530 and 620 nm. To further confirm cART-mediated lysosomal membrane permeabilization (LMP), cells were stained with GAL3 and LAMP2.