Acridine orange staining: AO/PI viability, AO/EB apoptosis and acidic organelles

Acridine orange is a small, cell-permeant cationic dye that binds nucleic acids in two ways, and the way it binds sets its colour. Slotted between the base pairs of double-stranded DNA, single dye molecules fluoresce green, near 525 nm. Stacked together on single-stranded RNA or denatured DNA, they fluoresce orange to red, near 650 nm. Both forms are excited by blue light, so a standard 488 nm laser or a FITC filter set works.

AO is also a weak base. In acidic compartments such as lysosomes it becomes protonated, cannot cross back out and builds up to concentrations where it stacks and fluoresces red. That one dye can report DNA, RNA, membrane integrity (with a partner dye) and acidic organelles is why so many different protocols carry its name.

Propidium iodide cannot cross an intact membrane. In a live cell only AO gets in and the nucleus is green; in a dead cell PI floods in, binds the DNA and its red signal overrides the green. Because both dyes stain nucleic acid, red blood cells, platelets and debris without a nucleus are not counted, which makes AO/PI more reliable than trypan blue for blood, bone marrow and tissue digests.

1. Mix the cell suspension with AO/PI staining solution, usually in equal volumes; follow the ratio your reagent gives.
2. Load the mixture into a counting slide or haemocytometer straight away.
3. Image or count within a few minutes; PI keeps entering dying cells, so a slide left standing reads lower.
4. Count green (live) and red (dead) cells.

Viability (%) = live cells ÷ (live + dead cells) × 100

In a direct comparison on bone marrow, AO/PI viability tracked known mixtures of live and heat-killed cells almost perfectly from 0 to 100% (r² 0.99), while trypan blue overestimated viability, most of all below 50%.

AO/EB works like AO/PI, but you read nuclear shape as well as colour. Apoptosis condenses and fragments chromatin before the membrane fails, so early apoptotic cells are still green but with bright, broken-up nuclei. The method can be run directly in a 96-well plate.

1. Culture and treat cells; for adherent lines, collect floating cells too, because apoptotic cells detach.
2. Add a small volume of combined AO/EB stock, commonly 100 µg/mL of each dye in PBS, to the cells.
3. View immediately under a fluorescence microscope with a blue excitation filter.
4. Score at least a few hundred cells across several fields, using the table below.

In live cells stained with AO alone, the cytoplasm and nucleus are green and the acidic vesicular organelles, lysosomes and autolysosomes, are red. When autophagy rises, the acidic compartment grows and so does the red signal. Measuring the ratio of red to green fluorescence per cell, rather than red alone, corrects for differences in dye uptake; this ratio tracked autophagy induced by starvation or rapamycin and fell when autophagy was blocked.

• Stain live cells only. Fixation collapses the pH gradient that traps the dye.
• Keep light exposure short. AO is phototoxic, and strong blue light can rupture dye-loaded lysosomes while you watch.
• Treat AO as a screen. It measures acidic volume, not autophagic flux, so confirm with LC3-II and p62 on a western blot using validated antibodies.

Staining shows what individual cells are doing; plate assays give a number per well for dose-response work. The MTT vs CCK-8 guide covers the plate side, and MTT troubleshooting explains readings that disagree with what you see under the microscope. AO/PI and AO/EB staining kits, viability assays and cell cycle kits are in the biochemical and cell-based assay range.