Acridine Orange | AAT Bioquest

Acridine orange

Acridine Orange is a cell-permeable nucleic acid stain that exhibits differential fluorescence—green for double-stranded DNA and red for RNA or single-stranded DNA—enabling simultaneous visualization of different nucleic acid types in live and fixed cells.

Differential Nucleic Acid Staining: Green fluorescence (490/520 nm) for dsDNA via intercalation, red fluorescence (460/640 nm) for RNA/ssDNA
Cell-Permeable Metachromatic Dye: Accumulates in acidic compartments including lysosomes at low pH
Weakly Basic Character: Neutral charge facilitates membrane diffusion for live cell imaging
Dual-Color Cell Viability: Combined with PI for simultaneous live/dead cell discrimination

Acridine Orange spectrum. Acridine Orange is a fluorescent compound with an excitation peak at 490 nm and an emission peak at 520 nm. Other spectra of interest include: Propidium iodide, 7-AAD (7-Aminoactinomycin D), and DAPI (4,6-Diamidino-2-phenylindole). Acridine Orange belongs to the following categories: Cell Cycle Assays, Nucleus, and Fluorescence Activated Cell Sorting (FACS).

Catalog	Size	Price	Quantity
17502	100 mg	Price

Overview

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

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.
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.
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.
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.
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.

Calculators

Common stock solution preparation

Table 1. Volume of Water needed to reconstitute specific mass of Acridine orange to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

	0.1 mg	0.5 mg	1 mg	5 mg	10 mg
1 mM	331.323 µL	1.657 mL	3.313 mL	16.566 mL	33.132 mL
5 mM	66.265 µL	331.323 µL	662.647 µL	3.313 mL	6.626 mL
10 mM	33.132 µL	165.662 µL	331.323 µL	1.657 mL	3.313 mL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)		Molecular weight		Volume (Calculate)		Concentration (Calculate)		Moles
mg	÷	301.82 g/mol	=	mL	x	mM	=	mmol

References

View all 56 references: Citation Explorer

Spectral studies of N-nonyl acridine orange in anionic, cationic and neutral surfactants

Authors:

Wiosetek-Reske AM, Wysocki S.

Journal:

Spectrochim Acta A Mol Biomol Spectrosc (2006): 1118

Re: The evaluation of micronucleus frequency by acridine orange fluorescent staining in peripheral blood of rats treated with lead acetate. (Mutagenesis, 20, 411-415, 2005)

Authors:

Celik A, Ogenler O, Comelekoglu U.

Journal:

Mutagenesis (2006): 267

Improvement of the acridine orange-protein-surfactant system for protein estimation based on aromatic ring stacking effect of sodium dodecyl benzene sulphonate

Authors:

Wang F, Yang J, Wu X, Wang X, Guo C, Jia Z.

Journal:

Luminescence (2006): 186

Vesicle disruption, plasma membrane bleb formation, and acute cell death caused by illumination with blue light in acridine orange-loaded malignant melanoma cells

Authors:

Hiruma H, Katakura T, Takenami T, Igawa S, Kanoh M, Fujimura T, Kawakami T.

Journal:

J Photochem Photobiol B. (2006)

The use of acridine orange base (AOB) as molecular probe to characterize nonaqueous AOT reverse micelles

Authors:

Falcone RD, Correa NM, Biasutti MA, Silber JJ.

Journal:

J Colloid Interface Sci (2006): 356

Physical properties

Molecular weight	301.82
Solvent	Water

Spectral properties

Absorbance (nm)	487
Extinction coefficient (cm ^-1 M ^-1)	27000
Excitation (nm)	490
Emission (nm)	520

Storage, safety and handling

Certificate of Origin	Download PDF
H-phrase	H303, H313, H340
Hazard symbol	T
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R68
Storage	Freeze (< -15 °C); Minimize light exposure
UNSPSC	41116134
CAS	65-61-2

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Page updated on September 21, 2025