Acridine Orange

Acridine orange is a cell permeable, nucleic acid dye that exhibits green fluorescence for dsDNA and red fluorescence for RNA or ssDNA.

Acridine orange is a derivative of acridine that was originally developed as a way to quickly label and identify bacteria. Over time, it has become established as a fluorophore for staining nucleic acids. Chemically, it has a neutral charge and is weakly basic, which facilitates its diffusion across cell membranes. As such, it can be used to stain live cells. Once in the cytoplasm, it tends to accumulate and be retained by acidic cellular compartments with low pH. Because of this, acridine orange has found use as a stain for lysosomes.

Nucleic acid staining

Given its weakly basic nature, acridine orange also binds to dsDNA, ssDNA and RNA, which are acidic. The binding to dsDNA is thought to occur by way of intercalation. This results in a low concentration of acridine orange around the nucleic acid, yielding a green fluorescence with an excitation peak at 490 nm and an emission peak at 520 nm. On the other hand, binding of acridine orange to RNA is thought to occur through electrostatic forces. This results in a higher concentration of the fluorophore and a red fluorescence, with an excitation peak at 460 nm and an emission peak at 640 nm.

Acridine orange and propidium iodide

Acridine orange is sometimes used in conjugation with propidium iodide to assess cell viability. Since acridine orange is cell permeable, it will stain live and dead cells. Propidium iodide, on the other hand, is membrane impermeant and will only stain dead cells. Used together, live and dead cells can be quantified.

Nonyl acridine orange

Nonyl acridine orange is a derivative of acridine orange that specifically accumulates in, and is used to study, mitochondria.

Further reading

1. Eriksson, I., Vainikka, L., Persson, H. L., & Öllinger, K. (2023). Real-time monitoring of lysosomal membrane permeabilization using acridine orange. Methods and Protocols, 6(4), 72.
2. McMaster, G. K., & Carmichael, G. G. (1977). Analysis of single-and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proceedings of the National Academy of Sciences, 74(11), 4835-4838.
3. Plemel, J. R., Caprariello, A. V., Keough, M. B., Henry, T. J., Tsutsui, S., Chu, T. H., ... & Stys, P. K. (2017). Unique spectral signatures of the nucleic acid dye acridine orange can distinguish cell death by apoptosis and necroptosis. Journal of Cell Biology, 216(4), 1163-1181.
4. Robbins, E., & Marcus, P. I. (1963). Dynamics of acridine orange-cell interaction: I. Interrelationships of acridine orange particles and cytoplasmic reddening. The Journal of cell biology, 18(2), 237-250.
5. Wolf, M. K., & Aronson, S. B. (1961). Growth, fluorescence and metachromasy of cells cultured in the presence of acridine orange. Journal of Histochemistry & Cytochemistry, 9(1), 22-29.