Acridine Orange (AO), a tricyclic heteroaromatic dye, is a metachromatic fluorescent dye. Capable of penetrating cell membranes, it stains nuclear DNA and RNA, and is therefore widely used for intracellular DNA and RNA detection.
AO binds to nucleic acids in two major modes:
1. Intercalative binding: AO intercalates between base pairs of double‑stranded nucleic acids. This mode mainly occurs with DNA, giving green fluorescence with an emission peak at 530 nm upon excitation.
2. Electrostatic attraction: Positively‑charged AO binds electrostatically to negatively‑charged phosphate groups of single‑stranded nucleic acids. This mode predominates for RNA binding, producing red fluorescence with an emission peak at 640 nm; weak binding yields orange‑yellow or orange‑red fluorescence.
Accordingly, AO exhibits green fluorescence when intercalated into double‑stranded DNA, and orange‑yellow or orange‑red fluorescence when bound to single‑stranded DNA or RNA.
Acridine Orange Staining Solution (1 mg/mL) is a stock solution and shall be diluted to an appropriate working concentration before use. AO is frequently applied in double staining together with EB. Since EB only labels dead cells with orange‑yellow fluorescence, normal cells, apoptotic cells and necrotic cells can be distinguished.
Instructions for Use
1. Harvest cells. Fix cells in advance for flow cytometry analysis. Wash cells once with PBS, count and adjust cell density to 10⁶ cells/mL.
2. Add adequate AO staining solution (1 mg/mL) into cell suspension to reach a final AO concentration of 8.5‑17 μg/mL, mix gently.
3. Incubate for 15‑20 min at room temperature in the dark. Mount aliquots onto glass slides with coverslips or proceed to flow cytometric analysis.
4. Observe under a fluorescence microscope (excitation filter: 488 nm; barrier filter: 515 nm), perform counting and image acquisition.
Precautions
1. AO staining solution (1 mg/mL) contains no permeabilizing agent and is seldom used alone.
2. AO‑EB double staining enables differentiation of normal, apoptotic and necrotic cells.
3. Better results can be obtained using a refrigerated centrifuge.
4. Minimize reagent exposure to intense light during manipulation.
5. Wear laboratory coat and disposable gloves for personal safety.
Staining Results
| Cell Type | Fluorescence Staining Characteristics | | Normal cells | Evenly stained yellow‑green fluorescence | | Apoptotic cells | Chromatin condensation and fragmented punctate nuclei, showing densely stained green granules of variable sizes |
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