Acridine orange (AO, catalog #6130) is an orange/red fluorescent chelating dye used to reveal lysosomes and nuclei in cultured cells. Due to its metachromatic properties, AO is commonly used in fluorescence microscopy and flow cytometry analysis of cellular physiology and cell cycle status. AO is a slightly cationic, lipophilic, weak base capable of permeating cell and organelle membrane structure. Although quite cell permeant in the neutral form, once protonated, this dye tends to become trapped on the low pH side of the membrane barrier leading to accumulation in acidic organelle structures. Proton pump driven lysosomal acidity generates a significant pH gradient resulting in the efficient concentration of AO within the lysosome organelles. This is sufficient to create intra-lysosomal concentrations leading to precipitation of AO into aggregated granules. These oligomeric structures exhibit a red shift (640 nm) compared to the monomeric AO that emits at 525 nm.

As AO exhibits a very broad emission range, one of several filter pairings on the fluorescence microscope can be used to view this stain. Using an excitation filter of 550 nm (540-560 nm) with a long pass >610 nm emission/barrier filter, lysosomes appear red. When illuminating with a blue light excitation filter (488 nm) and a green light emission/barrier filter (540-550 nm), lysosomes appear yellowish green instead of red. As this filter combination is very close to the maximum emission of AO, the slide may appear too bright. Excess AO may be removed by washing cells prior to viewing.

AO can be utilized in conjunction with a number of other staining techniques and fluorogenic substrates including ICT’s Magic Red®-DEVDase substrate (catalog #936) that detects caspase-3/7 activation in apoptotic cells. Because of the overlap in emissions, be wary of dual staining with other red stains as this will yield confusing results. Red dyes should be used separately.

AO may be used neat or diluted in diH2O, PBS, or media prior to pipetting into the cell suspension. Lysosomal structures can be visualized by staining with AO at 0.5-5.0 µM. This concentration range can be obtained by diluting ICT’s AO reagent stock (catalog #6130 at 1 mM) 1:2,000-1:200 (0.05-0.5% v/v) into the final cell suspension. For example, if using AO at 1.0 µM in the final cell suspension, it must be diluted 1:1,000. First dilute it 1:100 in PBS; e.g., put 10 µL AO into 990 µL PBS. Pipette the diluted AO into the cell suspension at approximately 1:10; e.g., put 50 µL diluted AO into 450 µL cell suspension. Always protect AO from bright light.