Acridine Orange Fluorescent Staining Solution

About this product

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.

Specifications

Catalog number

6130

Size / count

0.5 mL

Dry ice

No

Hazardous

No

Discontinued

Yes

Label

ICT

Cell Type

Jurkat; MCF-7

Type

Cell Viabiity

Detection Method

Flow cytometry, Fluorescence microscope

Wavelength

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)

Assay Protocol

Dilute 10X PBS 1:10, let the solution stir for 15 minutes, and use. As PBS is concentrated, crystalline precipitates may form in the bottle, especially when refrigerated. If this happens, gently warm or mix the buffer until all crystals are dissolved. Do not boil.

Concentration

1 mM, 266 µg/mL

pH

pH: 5.0 + 0.5

Shipping Conditions

Ships overnight (domestic), International Priority Shipping

Storage Temperature

2-8°C

Calculated Molecular Weight

301.82

Cellular Imaging & Detection

Cellular Imaging

Target Description

Acridine Orange (AO) is a slightly cationic, lipophilic, fluorochrome stain capable of permeating cell and organelle membrane structure. 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., , 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. Proton pump-driven lysosomal acidity generates a significant pH gradient resulting in the efficient concentration of AO within the lysosome organelles. 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., , 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 and the Magic Red Caspase 3/7 that detects caspase 3/7 activation in apoptotic cells.