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).
- 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
- Han, J., et al. Fluorescent indicators for intracellular pH. Chem Rev 110, 2709-2728 (2010).
- 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).
- Darzynkiewicz, z. Flow cytometric methods for RNA content analysis. Methods 2(3), 200-206, (1991).
- 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).
- Kobayashi, Y., et al. Mechanism of apoptosis induced by a lysosomotropic agent, L-leucyl-L-leucine methyl ester. Apoptosis 4, 357-362 (1999).
- Traganos, F., et al. Lysosomal proton pump activity: Supravital cell staining with acridine orange differentiates leukocyte subpopulations. Method Cell Biol. 41, 185-194, (1994).
- 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).
- 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).
|Best For:||Cellular imaging and lysosomal staining|
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).
|Storage:||2°-8° C, Ships Overnight (Domestic), International Priority Shipping|