Resazurin

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 (Trek Diagnostic Systems, Inc), and now also available under other names such as AB assay, Vybrant (Molecular Probes) and UptiBlue (Interchim).

Resazurin based assays show excellent correlation to reference viability assays such as formazan-based assays (MTT/XTT) 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 In antimicrobial assays, resazurin is commonly utilized to assess the minimum inhibitory concentration (MIC) or minimum bactericidal concentration (MBC) of antimicrobial agents.

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.

|Image:Resazurin assay for mammalian cell viability.JPG|Resazurin as a colorimetric assay for cell viability |Image:Resazurin assay - Resazurin does not fluoresce.JPG|Resazurin used as a fluorescent assay for cell viability - Resazurin does not fluoresce when exposed to green light. |Image:Resazurin assay - Resorufin fluoresces.JPG|Resazurin as a fluorescent assay for cell viability - Resorufin fluoresces when exposed to green light.

Resazurin reduction test

Resazurin can be used as one of a series of rapid tests to determine the quality of a milk sample. In this test, resazurin is added as a violet redox dye which turns mauvish-pink due to conversion to resorufin and then to colourless dihydroresorufin. This happens due to lowering of the oxidation-reduction potential in the milk sample caused by presence of bacteria which utilize available oxygen present in the milk for aerobic respiration. The rate of the colour change is used as an index for the number of bacteria present in the milk sample.

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(IV) oxide:

Treatment of the crude reaction product with excess sodium carbonate yields the sodium salt 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 (also known as Amplex Red), structurally related to resazurin, reacts with H2O2 in a 1:1 stoichiometry to produce the same by-product resorufin (used in many assays combining for example horseradish peroxidase (HRP), or NADH, NADPH using enzymes).

7-ethoxyresorufin, a compound used as the substrate in the measurement of cytochrome P450 (CYP1A1) induction using the ethoxyresorufin-O-deethylase (EROD) 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(9H)-one (DDAO dye), a fluorescent dye used for oligonucleotide labeling.

References

References

1. "Resazurin | C12H7NO4 | ChemSpider".
2. (2002). "The Excited-State Interaction of Resazurin and Resorufin with Aminesin Aqueous Solutions. Photophysics and Photochemical Reaction". Photochemistry and Photobiology.
3. (2009). "Resazurin as a "smart" tracer for quantifying metabolically active transient storage in stream ecosystems". Journal of Geophysical Research: Biogeosciences.
4. "Resazurin, sodium salt".
5. Chen, J. L., Steele, T. W., & Stuckey, D. C. (2015). Modeling and application of a rapid fluorescence-based assay for biotoxicity in anaerobic digestion. Environmental science & technology, 49(22), 13463-13471.http://pubs.acs.org/doi/10.1021/acs.est.5b03050
6. (2009). "Quantification of dichromatism: A characteristic of color in transparent materials". Journal of the Optical Society of America A.
7. (1929). "Combined assays for lactose and galactose by enzymatic reactions". Milchw. Forsch.
8. (2005). "Resazurin assay of radiation response in cultured cells". British Journal of Radiology.
9. (January 2023). "Cytotoxic Activity of Herbal Medicines as Assessed in Vitro: A Review". Chemistry & Biodiversity.
10. [http://www.interchim.fr/ft/6/66941f.pdf UptiBlue viable cell assay] technical manual
11. (2012). "Synergy Study of the Inhibitory Potential of Red Wine Polyphenols on Vascular Smooth Muscle Cell Proliferation". Planta Medica.
12. (2024). "Methods for screening and evaluation of antimicrobial activity: A review of protocols, advantages, and limitations". European Journal of Microbiology and Immunology.
13. (2012). "Measuring aerobic respiration in stream ecosystems using the resazurin-resorufin system". Journal of Geophysical Research.
14. Tortorello, Mary Lou, Batt, Carl A.. (2 April 2014). "Encyclopedia of food microbiology". Academic Press.
15. (1984). "An Enzymatic Method for the Analysis of Formate in Human Plasma". Journal of Analytical Toxicology.
16. (1983). "Effect of protein on the determination of total bile acids in serum". Clinical Chemistry.
17. (1977). "Fluorimetric assay of tobacco leaf dehydrogenases with resazurin". Biochimica et Biophysica Acta (BBA) - Enzymology.
18. (1980). "Stoichiometry of the nadh-oxidoreductase reaction for dehydrogenase determinations". Clinica Chimica Acta.
19. (1980). "Fluorometric and colorimetric enzymic determination of triglycerides (triacylglycerols) in serum". Clinical Chemistry.
20. (1999). "Microplate-based fluorometric methods for the enzymatic determination of l-glutamate: application in measuring l-glutamate in food samples". Analytica Chimica Acta.
21. (2014). "Methods for assessing biochemical oxygen demand (BOD): A review". Water Research.
22. (2009). "Resazurin as a 'smart' tracer for quantifying metabolically active transient storage in stream ecosystems". Journal of Geophysical Research.
23. "Dyestuffs suitable for use as indicators".
24. (1997). "A stable nonfluorescent derivative of resorufin for the fluorometric determination of trace hydrogen peroxide: Applications in detecting the activity of phagocyte NADPH oxidase and other oxidases". Anal Biochem.
25. Mohammadi-Bardbori, Afshin. (2014). "Assay for quantitative determination of CYP1A1 enzyme activity using 7-Ethoxyresorufin as standard substrate (EROD assay)". Research Square.
26. (2005). "Cytochrome P450 Protocols".
27. (2010). "Extracellular DNA is abundant and important for microcolony strength in mixed microbial biofilms". Environmental Microbiology.