Acridine Orange Staining Solution

Catalog Number: 6130 (0.5 mL)

Availability: In stock

$20.00
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Quick Overview

Acridine Orange is used for fluorescence microscopy and flow cytometry analysis of cellular physiology and cell cycle status. This cell-permeant cellular stain can be utilized in conjunction with a number of other staining techniques and fluorogenic substrates including the Magic Red line of fluorogenic protease substrates. Acridine Orange is a useful bacteria stain for the fluorescent microscopic examination of microorganisms.


Ordering information:
Catalog number: 6130

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Acridine Orange (AO) staining of MCF-7 cells.  Cells were stained with 0.5 uM AO in PBS for 30 minutes, washed twice in PBS, and photographed using blue light excitation (480 nm) with 540-550 nm emission (Dr. Zbigniew Darzynkiewicz).

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  • Acridine Orange Staining Solution
  • Acridine Orange (AO) staining of MCF-7 cells.  Cells were stained with 0.5 uM AO in PBS for 30 minutes, washed twice in PBS, and photographed using blue light excitation (480 nm) with 540-550 nm emission (Dr. Zbigniew Darzynkiewicz).
  • Acridine Orange (AO) staining of MCF-7 cells. Cells were stained with 0.5 uM AO in PBS for 30 minutes, washed twice in PBS, and photographed using green light excitation (540 nm) with long pass > 640 emission (Dr. Zbigniew Darzynkiewicz).

Acridine Orange (AO) is a slightly cationic, lipophilic, fluorochrome stain capable of permeating cell and organelle membrane structure (1). Although quite cell permeant in the neutral form, once protonated, these dyes tend to become trapped on the low pH side of the membrane barrier leading to their accumulation in acidic organelle structures, such as lysosomes (2-6).

Acridine Orange (AO), due to its metachromatic properties, is commonly used in fluorescence microscopy and flow cytometry analysis of cellular physiology and cell cycle status, including the fluorescent microscopic examination of microorganisms (4-5). Proton pump-driven lysosomal acidity generates a significant pH gradient resulting in the efficient concentration of AO within the lysosome organelles (6). The effectiveness of this AO concentration process is sufficient to create intra-lysosomal concentrations leading to precipitation of the AO into aggregated granules. These oligomeric structures exhibit a red shift (640 nm) compared to the monomeric AO that emits at 525 nm (7).

Acridine Orange (AO) can be utilized in conjunction with a number of other staining techniques and fluorogenic substrates, including the Magic Red Cathepsin B substrate (reagent (MR-RR)2, cat# 937 and 938) and the Magic Red Caspase 3/7 (DEVDases) substrate ((MR-DEVD)2, cat# 935 and 936) that detects caspase 3/7 activation in apoptotic cells (8).

Specifications:

  • 0.5 mL at 1 mM (266 μg/mL)
  • CAS number 65-61-2
  • Chemical name 3,6-acridinediamine, N, N, N’, N’-tetramethylmonohydrochloride
  • Molecular formula C17H20ClN3
  • Molecular weight 301.82
  • Pale red/orange liquid
  • pH 5.0 ± 0.5

References

  1. Han, J., et al. Fluorescent indicators for intracellular pH.  Chem Rev 110, 2709-2728 (2010).
  2. Darzynkiewicz, Z., et al., Differential staining of DNA and RNA. In: Robinson, J.P. et al. editors.  Current Protocols in Cytometry. New Jersey, USA: John Wiley & Sons; Unit 7.3, (2004).
  3. Darzynkiewicz, z. Flow cytometric methods for RNA content analysis.  Methods 2(3), 200-206, (1991).
  4. Darzynkiewicz, Z., et al., Analysis of DNA denaturation. In: Robinson, J.P. et al. editors.  Current Protocols in Cytometry. New Jersey, USA: John Wiley & Sons; Unit 7.8, (1998).
  5. Kobayashi, Y., et al. Mechanism of apoptosis induced by a lysosomotropic agent, L-leucyl-L-leucine methyl ester.  Apoptosis 4, 357-362 (1999).
  6. Traganos, F., et al.  Lysosomal proton pump activity: Supravital cell staining with acridine orange differentiates leukocyte subpopulations. Method Cell Biol. 41, 185-194, (1994).
  7. Darzynkiewicz, Z., et al. Acridine orange: A versatile probe of nucleic acids and other cell constituents.  In: Flow Cytometry and Sorting. (M.R. Melamed et al. eds. New York, USA: Wiley-Liss 291-314, (1990).
  8. Lee, B., et al. DEVDase detection in apoptotic cells using the cell permeant fluorogenic substrate, (z-DEVD)2-cresyl violet. BioTechniques 35(5), 1080-1085, (2003).
Product Datasheet:

Acridine Orange, 0.5 mL vial

Best For: Cellular imaging and lysosomal staining
To Use:

AO may be used neat or diluted in diH2O, PBS, or media. ICT’s catalog #6130 is supplied as 0.5 mL liquid at 1 mM (266 μg/mL). 

  1. 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 (0.05-0.5% v/v). For example, if using AO at 1.0 μM in the final cell suspension, it must be diluted 1:1,000: 2a. First dilute it 1:100 in PBS or diH2O; e.g., put 10 μL AO into 990 μL PBS or diH2O. 2b. 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:
    4a. 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.
    4b. 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.

 

Contains:
  • 0.5 mL at 1 mM (266 μg/mL)
  • Molecular weight 301.82
  • Pale red/orange liquid
  • pH 5.0 ± 0.5
Storage: 2°-8° C, Ships Overnight (Domestic), International Priority Shipping
MSDS:

Acridine Orange Stain MSDS