Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity
Open accessAbstract
We show here the identity of Alamar Blue as resazurin. The ‘resazurin reduction test’ has been used for about 50 years to monitor bacterial and yeast contamination of milk, and also for assessing semen quality. Resazurin (blue and nonfluorescent) is reduced to resorufin (pink and highly fluorescent) which is further reduced to hydroresorufin (uncoloured and nonfluorescent). It is still not known how this reduction occurs, intracellularly via enzyme activity or in the medium as a chemical reaction, although the reduced fluorescent form of Alamar Blue was found in the cytoplasm and of living cells nucleus of dead cells. Recently, the dye has gained popularity as a very simple and versatile way of measuring cell proliferation and cytotoxicity. This dye presents numerous advantages over other cytotoxicity or proliferation tests but we observed several drawbacks to the routine use of Alamar Blue. Tests with several toxicants in different cell lines and rat primary hepatocytes have shown accumulation of the fluorescent product of Alamar Blue in the medium which could lead to an overestimation of cell population. Also, the extensive reduction of Alamar Blue by metabolically active cells led to a final nonfluorescent product, and hence an underestimation of cellular activity.
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Resazurin, a nonproprietary product, has been identified as the main component of Alamar blue.
“Recently, resazurin, a nonproprietary product, has been identified as the main component of Alamar blue (34, 43).”
From Resazurin Microtiter Assay Plate Testing of <i>Mycobacterium tuberculosis</i> Susceptibilities to Second-Line Drugs: Rapid, Simple, and Inexpensive Method (2003) · cites this paper as [34]
Resazurin has been recently identified as the main component of Alamar blue.
“Since resazurin has been recently identified as the main component of Alamar blue (34, 43), we recently developed a resazurin microtiter assay (REMA) plate (37) for detecting MDR TB and demonstrated a very good correlation between results by this method and the proportion method (PM).”
From Resazurin Microtiter Assay Plate Testing of <i>Mycobacterium tuberculosis</i> Susceptibilities to Second-Line Drugs: Rapid, Simple, and Inexpensive Method (2003) · cites this paper as [34]
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- Seglen, P.O. (1993) Isolation of hepatocytes by collagenase perfusion. In In Vitro Biological Systems (Tyson, C.A. & Frazier, J.M., eds), pp. 231±243. Academic Press, San Diego.
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- Authors
- O'Brien, J., Wilson, I., Orton, T., & Pognan, F.
- Affiliations
- AstraZeneca (United Kingdom) (GB)
- ORCID
- Wilson, Ian
- Venue
- European Journal of Biochemistry
- Publisher
- Wiley
- Published
- 2000-09
- Volume
- 267
- Issue
- 17
- Pages
- 5421-5426
- ISSN
- 0014-2956 (ISSN-L)
- 1432-1033
- OpenAlex
- W2154385453
- Cited by
- 3,446
- References
- 0
- Citations by year
- 2026: 119 · 2025: 153 · 2024: 168 · 2023: 164 · 2022: 167 · 2021: 183 · 2020: 215 · 2019: 196 · 2018: 207 · 2017: 224 · 2016: 204 · 2015: 186 · 2014: 195 · 2013: 179 · 2012: 164
- FWCI
- 34.14
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- European Journal of Biochemistry (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Health Sciences › Veterinary › Small Animals › Animal testing and alternatives
- Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Toxicology › Bioactive Compounds and Antitumor Agents
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Cell Image Analysis Techniques
- Keywords
- Alamar Blue, resazurin, resorufin, cytotoxicity assay, cell proliferation, dye reduction
- MeSH terms
- Animals, Cell Survival, Culture Media, Fluorescent Dyes, Male, Magnetic Resonance Spectroscopy, Oxazines, Mass Spectrometry, Xanthenes, Rats, Wistar, Rats
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