Acridine Orange Fluorescent Staining Solution

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For fluorescence microscopy and flow cytometry analysis of cellular physiology and cell cycle status. It may be used in conjunction with other stains / fluorogenic substrates including our Magic Red® products. Can also be used as a bacterial stain.

Figure 1. Normal Jurkat cells stained with Acridine Orange (AO) show orange lysosomal staining. Cells were stained with 5 µM AO in PBS for 60 minutes at 37°C. Photomicrographs were taken using a Nikon Eclipse E800 photomicroscope using a 460-500 nm excitation filter and a 505-560 nm emission / barrier filter set at 300X. Photo B shows the corresponding DIC image of the cells in A (AO appears faintly). Data courtesy of Dr. Zbigniew Darzynkiewicz (Brander Cancer Research Institute, New York, NY).
Figure 2. MCF-7 cells were treated with camptothecin to induce apoptosis (cat. 6210), stained with AO in PBS, washed, and then photographed with an epifluorescence microscope at 40X using either a blue light excitation (492 nm) with a 540- 550 nm emission filter (A, lysosomes appear yellowish green), or green light excitation (540 nm) with a long pass >640 nm barrier filter (B, lysosomes appear red). Data courtesy of Dr. Zbigniew Darzynkiewicz (Brander Cancer Research Center Institute, New York, NY).
Figure 1. Normal Jurkat cells stained with Acridine Orange (AO) show orange lysosomal staining. Cells were stained with 5 µM AO in PBS for 60 minutes at 37°C. Photomicrographs were taken using a Nikon Eclipse E800 photomicroscope using a 460-500 nm excitation filter and a 505-560 nm emission / barrier filter set at 300X. Photo B shows the corresponding DIC image of the cells in A (AO appears faintly). Data courtesy of Dr. Zbigniew Darzynkiewicz (Brander Cancer Research Institute, New York, NY).
Figure 1. Normal Jurkat cells stained with Acridine Orange (AO) show orange lysosomal staining. Cells were stained with 5 µM AO in PBS for 60 minutes at 37°C. Photomicrographs were taken using a Nikon Eclipse E800 photomicroscope using a 460-500 nm excitation filter and a 505-560 nm emission / barrier filter set at 300X. Photo B shows the corresponding DIC image of the cells in A (AO appears faintly). Data courtesy of Dr. Zbigniew Darzynkiewicz (Brander Cancer Research Institute, New York, NY).
Figure 2. MCF-7 cells were treated with camptothecin to induce apoptosis (cat. 6210), stained with AO in PBS, washed, and then photographed with an epifluorescence microscope at 40X using either a blue light excitation (492 nm) with a 540- 550 nm emission filter (A, lysosomes appear yellowish green), or green light excitation (540 nm) with a long pass >640 nm barrier filter (B, lysosomes appear red). Data courtesy of Dr. Zbigniew Darzynkiewicz (Brander Cancer Research Center Institute, New York, NY).

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Data Sheet Safety Data Sheet

SKU: 6130

Size: 0.5 mL
Price:
$22.75

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.

492 nm / 525 nm (monomeric form), 502 nm / 520-524 nm (Aggregated or DNA complexed form), 457 nm / 630-644 nm (Aggregated or RNA complexed form)
Flow cytometry, Fluorescence microscope
2-8°C
Domestic: Overnight Delivery; International: Priority Shipping
4.0 – 5.5
1 mM, 266 µg/mL
United States
  1. AO may be used neat or diluted in diH2O, PBS, or media. ICT’s AO is supplied at 0.5 mL liquid at 1 mM (266 µg/mL). 2. Add AO to the cell sample media at 0.5-5 µM, equal to a final dilution of 1:2,000 – 1:200 in the cells (.05-.5% v/v). 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 or diH2O; e.g., put 10 µL AO into 990 µL PBS or diH2O. Pipette the diluted AO into the cell suspension at approximately 1:10; e.g., put 50 µL diluted AO into 450 µL cell suspension.
  2. Incubate 15-30 minutes at 37°C.
  3. Wash cells if reagent is too bright.
  4. Analyze with fluorescence. Lysosomes will appear yellowish green by illuminating cells with a blue light (488 nm) excitation filter and a green light (540-550 nm) emission/barrier filter. Alternatively, lysosomes will appear red when using an excitation filter of 550 nm (540-560 nm) and a long pass >610 nm emission/barrier filter.

Product Specific References

PMID Publication
38441406Vinik, Y, et al. 2024. Programming a Ferroptosis-to-Apoptosis Transition Landscape Revealed Ferroptosis Biomarkers and Repressors for Cancer Therapy. Advanced science (Weinheim, Baden-Wurttemberg, Germany), e2307263.
35087718Matthaiou, EI, et al. 2022. TEM1-targeting PEGylated PLGA shikonin nanoformulation for immunomodulation and eradication of ovarian cancer. BioImpacts: BI, 65-86.
35429112Duarte, C, et al. 2022. Crosstalk between dihydroceramides produced by Porphyromonas gingivalis and host lysosomal cathepsin B in the promotion of osteoclastogenesis. Journal of cellular and molecular medicine .
35654241Kim, NY, et al. 2022. Tanshinone IIA exerts autophagic cell death through down-regulation of β-catenin in renal cell carcinoma cells. Biochimie.

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