Resazurin is a phenoxazinedye that is weakly fluorescent, nontoxic, cell-permeable, and redox‐sensitive. Resazurin has a blue to purplecolor and is used in microbiological, cellular, and enzymatic assays because it can be irreversibly reduced to the pink-colored and highly fluorescent resorufin. At circum-neutral pH, resorufin can be detected by visual observation of its pink color or by fluorimetry, with an excitation maximum at 530-570 nm and an emission maximum at 580-590 nm. When a solution containing resorufin is submitted to reducing conditions, almost all resorufin is reversibly reduced to the translucid non-fluorescent dihydroresorufin and the solution becomes translucid. When the Eh of this same solution is increased, dihydroresorufin is oxidized back to resorufin, and this reversible reaction can be used to monitor if the redox potential of a culture medium remains at a sufficiently low level for anaerobic organisms. Resazurin solution has one of the highest values known of Kreft's dichromaticity index. This means that it has a large change in perceived color hue when the thickness or concentration of observed sample increases or decreases. Usually, resazurin is available commercially as the sodium salt.
Cell viability applications
Resazurin is reduced to resorufin by aerobic respiration of metabolically active cells, and it can be used as an indicator of cell viability. It was first used to quantify bacterial content in milk by Pesch and Simmert in 1929. It can be used to detect the presence of viable cells in mammalian cell cultures. It was introduced commercially initially under Alamar Blue trademark, and now also available under other names such as AB assay, Vybrant and UptiBlue. Resazurin based assays show excellent correlation to reference viability assays such as formazan-based assays and tritiated thymidine based techniques. The low toxicity makes it suitable for longer studies, and it has been applied for animal cells, bacteria, and fungi for cell culture assays such as cell counting, cell survival, and cell proliferation.. To take the place of a standard live/dead assay, resazurin also be multiplexed with chemiluminescent assays, such as cytokine assays, caspase assays to measure apoptosis, or reporter assays to measure a gene or a protein expression. The irreversible reaction of resazurin to resorufin is proportional to aerobic respiration.
Other applications
Resazurin is effectively reduced in mitochondria, making it useful also to assess mitochondrial metabolic activity. Usually, in the presence of NADPH dehydrogenase or NADH dehydrogenase as the enzyme, NADPH or NADH is the reductant that converts resazurin to resorufin. Hence the resazurin/diaphorase/NADPH system can be used to detect NADH, NADPH, or diaphorase level, and any biochemical or enzyme activity that is involved in a biochemical reaction generating NADH or NADPH. Resazurin can be used to assay L-Glutamate, achieving a sensitivity of 2.0 pmol per well in a 96 well plate. Resazurin can also be used to measure the aerobic biodegradation of organic matter found in effluents. Resazurin is used to measure the amount of aerobic respiration in streams Since most aerobic respiration occurs in the stream bed, the conversion of resazurin to resorufin is also a measure of the amount of exchange between the water column and the stream bed.
Synthesis
Resazurin is prepared by acid-catalyzed condensation between resorcinol and 4-nitrosoresorcinol followed by oxidation of the intermediate with manganese oxide: Treatment of the crude reaction product with excess sodium carbonate yields the sodiumsalt of resazurin, which is typically the commercial form of the dye. Running the condensation step in alcohols is possible but results in lower yields of the product; in pure water or acetic acid, the reaction does not proceed satisfactorily.
Related dyes
10-acetyl-3,7-dihydroxyphenoxazine, structurally related to resazurin, reacts with H2O2 in a 1:1 stoichiometry to produce the same by-product resorufin. 7-ethoxyresorufin, is a compound used as the substrate in the measurement of cytochrome P450 induction using the ethoxyresorufin-O-deethylase assay system in cell culture and environmental samples, produced in response to exposure to aryl hydrocarbons. The compound is catalysed by the enzyme to produce the same fluorescent product, resorufin. 1,3-dichloro-7-hydroxy-9,9-dimethylacridin-2-one, fluorescent dye used for oligonucleotide labeling.
