Resazurin (7-Hydroxy-3''H''-phenoxazin-3-one 10-oxide) is a
phenoxazine
Phenoxazine is a heterocyclic compound. The structure of phenoxazine consists of an oxazine fused to two benzene rings. It occurs as the central core of a number of naturally occurring chemical compounds such as dactinomycin and litmus. The dyes ...
dye
Juan de Guillebon, better known by his stage name DyE, is a French musician. He is known for the music video of the single "Fantasy
Fantasy is a genre of speculative fiction that involves supernatural or Magic (supernatural), magical ele ...
that is weakly
fluorescent
Fluorescence is one of two kinds of photoluminescence, the emission of light by a substance that has absorbed light or other electromagnetic radiation. When exposed to ultraviolet radiation, many substances will glow (fluoresce) with color ...
,
nontoxic
Toxicity is the degree to which a chemical substance or a particular mixture of substances can damage an organism. Toxicity can refer to the effect on a whole organism, such as an animal, bacterium, or plant, as well as the effect on a substr ...
, cell-permeable, and redox‐sensitive. Resazurin has a
blue
Blue is one of the three primary colours in the RYB color model, RYB colour model (traditional colour theory), as well as in the RGB color model, RGB (additive) colour model. It lies between Violet (color), violet and cyan on the optical spe ...
to
purple
Purple is a color similar in appearance to violet light. In the RYB color model historically used in the arts, purple is a secondary color created by combining red and blue pigments. In the CMYK color model used in modern printing, purple is ...
color
Color (or colour in English in the Commonwealth of Nations, Commonwealth English; American and British English spelling differences#-our, -or, see spelling differences) is the visual perception based on the electromagnetic spectrum. Though co ...
above
pH 6.5 and an orange color below pH 3.8. It is used in
microbiological
Microbiology () is the scientific study of microorganisms, those being of unicellular (single-celled), multicellular (consisting of complex cells), or acellular (lacking cells). Microbiology encompasses numerous sub-disciplines including virol ...
,
cellular, and
enzymatic
An enzyme () is a protein that acts as a biological catalyst by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as produc ...
assays because it can be irreversibly
reduced
Reduction, reduced, or reduce may refer to:
Science and technology Chemistry
* Reduction (chemistry), part of a reduction-oxidation (redox) reaction in which atoms have their oxidation state changed.
** Organic redox reaction, a redox reacti ...
to the
pink
Pink is a pale tint of red, the color of the Dianthus plumarius, pink flower. It was first used as a color name in the late 17th century. According to surveys in Europe and the United States, pink is the color most often associated with charm, p ...
-colored and highly fluorescent
resorufin (7-Hydroxy-3''H''-phenoxazin-3-one). 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 (E
h < -110 mV), almost all resorufin is reversibly reduced to the
translucid non-fluorescent
dihydroresorufin (also known as hydroresorufin) and the solution becomes translucid (the
redox potential
Redox potential (also known as oxidation / reduction potential, ''ORP'', ''pe'', ''E_'', or E_) is a measure of the tendency of a chemical species to acquire electrons from or lose electrons to an electrode and thereby be reduced or oxidised respe ...
of the resorufin/dihydroresorufin pair is -51 mV vs.
standard hydrogen electrode
In electrochemistry, the standard hydrogen electrode (abbreviated SHE), is a redox electrode which forms the basis of the thermodynamic scale of oxidation-reduction potentials. Its absolute electrode potential is estimated to be at 25 ° ...
at pH 7.0). When the E
h 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
A growth medium or culture medium is a solid, liquid, or semi-solid designed to support the growth of a population of microorganisms or cells via the process of cell proliferation or small plants like the moss '' Physcomitrella patens''. Diffe ...
remains at a sufficiently low level for
anaerobic organism
An anaerobic organism or anaerobe is any organism that does not require oxygen, molecular oxygen for growth. It may react negatively or even die if free oxygen is present. In contrast, an aerobic organism (aerobe) is an organism that requires an o ...
s.
Resazurin solution has one of the highest values known of
Kreft's dichromaticity index
Kreft's dichromaticity index (DI) is a measure for quantification of dichromatism. It is defined as the difference in hue angle (Δhab) between the color of the sample at the dilution, where the chroma (color saturation) is maximal, and the color ...
. This means that it has a large change in perceived color
hue
In color theory, hue is one of the properties (called color appearance parameters) of a color, defined in the CIECAM02 model as "the degree to which a stimulus can be described as similar to or different from stimuli that are described as ...
when the thickness or concentration of observed sample increases or decreases.
Usually, resazurin is available commercially as the
sodium
Sodium is a chemical element; it has Symbol (chemistry), symbol Na (from Neo-Latin ) and atomic number 11. It is a soft, silvery-white, highly reactive metal. Sodium is an alkali metal, being in group 1 element, group 1 of the peri ...
salt
In common usage, salt is a mineral composed primarily of sodium chloride (NaCl). When used in food, especially in granulated form, it is more formally called table salt. In the form of a natural crystalline mineral, salt is also known as r ...
.
Cell viability applications
Resazurin is reduced to resorufin by
aerobic respiration
Cellular respiration is the process of oxidizing biological fuels using an inorganic electron acceptor, such as oxygen, to drive production of adenosine triphosphate (ATP), which stores chemical energy in a biologically accessible form. Cellu ...
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 Molecular Probes was a biotechnology company located in Eugene, Oregon specializing in fluorescence. The company was founded in 1975 by Richard and Rosaria Haugland in their kitchen in Minnesota, then moved briefly to Texas and finally to Oregon in ...
) and UptiBlue (
Interchim
Interchim is a privately owned French language, French company specialized in manufacturing and distribution of reagents, consumables and dedicated instruments for the Research and development, R&D and industry laboratory in the fields of Fine ch ...
).
Resazurin based assays show excellent correlation to reference viability assays such as
formazan
The formazans are compounds of the general formula -N=N-C(R')=N-NH-R" formally derivatives of formazan 2NN=CHN=NH unknown in free form.
Formazan dyes are artificial Chromogen, chromogenic products obtained by reduction of tetrazolium salts ...
-based assays (
MTT/XTT) and tritiated thymidine based techniques.
[UptiBlue viable cell assay]
technical manual The low toxicity makes it suitable for longer studies, and it has been applied for animal cells, bacteria, and fungi
for
cell culture assays
A cell culture assay is any method used to assess the cytotoxicity of a material. This refers to the ''in vitro'' assessment of a material to determine whether it releases toxic chemicals in the cell. It also determines if the quantity is suffici ...
such as cell counting, cell survival, and
cell proliferation
Cell proliferation is the process by which ''a cell grows and divides to produce two daughter cells''. Cell proliferation leads to an exponential increase in cell number and is therefore a rapid mechanism of tissue growth. Cell proliferation ...
. In antimicrobial assays, resazurin is commonly utilized to assess the
minimum inhibitory concentration
In microbiology, the minimum inhibitory concentration (MIC) is the lowest concentration of a chemical, usually a drug, which prevents visible ''in vitro'' cell growth, growth of bacteria or Fungus, fungi. MIC testing is performed in both diagnosti ...
(MIC) or
minimum bactericidal concentration The minimum bactericidal concentration (MBC) is the lowest concentration of an antibacterial agent required to kill a particular bacterium. It can be determined from broth dilution minimum inhibitory concentration (MIC) tests by subculturing to ag ...
(MBC) of
antimicrobial
An antimicrobial is an agent that kills microorganisms (microbicide) or stops their growth (bacteriostatic agent). Antimicrobial medicines can be grouped according to the microorganisms they are used to treat. For example, antibiotics are used aga ...
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.
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
Cellular respiration is the process of oxidizing biological fuels using an inorganic electron acceptor, such as oxygen, to drive production of adenosine triphosphate (ATP), which stores chemical energy in a biologically accessible form. Cellu ...
. 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
A mitochondrion () is an organelle found in the cells of most eukaryotes, such as animals, plants and fungi. Mitochondria have a double membrane structure and use aerobic respiration to generate adenosine triphosphate (ATP), which is us ...
, making it useful also to assess
mitochondria
A mitochondrion () is an organelle found in the cells of most eukaryotes, such as animals, plants and fungi. Mitochondria have a double membrane structure and use aerobic respiration to generate adenosine triphosphate (ATP), which is us ...
l
metabolic
Metabolism (, from ''metabolē'', "change") is the set of life-sustaining chemical reactions in organisms. The three main functions of metabolism are: the conversion of the energy in food to energy available to run cellular processes; the ...
activity.
Usually, in the presence of
NADPH dehydrogenase or
NADH dehydrogenase
NADH dehydrogenase is an enzyme that converts nicotinamide adenine dinucleotide (NAD) from its reduced form (NADH) to its oxidized form (NAD+). Members of the NADH dehydrogenase family and analogues are commonly systematically named using the f ...
as the enzyme,
NADPH
Nicotinamide adenine dinucleotide phosphate, abbreviated NADP or, in older notation, TPN (triphosphopyridine nucleotide), is a cofactor used in anabolic reactions, such as the Calvin cycle and lipid and nucleic acid syntheses, which require N ...
or
NADH
Nicotinamide adenine dinucleotide (NAD) is a coenzyme central to metabolism. Found in all living cells, NAD is called a dinucleotide because it consists of two nucleotides joined through their phosphate groups. One nucleotide contains an ade ...
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
Glutamic acid (symbol Glu or E; known as glutamate in its anionic form) is an α-amino acid that is used by almost all living beings in the biosynthesis of proteins. It is a non-essential nutrient for humans, meaning that the human body can s ...
, 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
Resorcinol (or resorcin) is a phenolic compound. It is an organic compound with the formula C6H4(OH)2. It is one of three isomeric benzenediols, the 1,3-isomer (or ''meta- (chemistry), meta''-isomer). Resorcinol crystallizes from benzene as co ...
and 4-nitrosoresorcinol followed by oxidation of the intermediate with
manganese(IV) oxide:
Treatment of the crude reaction product with excess
sodium carbonate
Sodium carbonate (also known as washing soda, soda ash, sal soda, and soda crystals) is the inorganic compound with the formula and its various hydrates. All forms are white, odourless, water-soluble salts that yield alkaline solutions in water ...
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 H
2O
2 in a 1:1 stoichiometry to produce the same by-product resorufin (used in many assays combining for example
horseradish peroxidase
The enzyme horseradish peroxidase (HRP), found in the roots of horseradish, is used extensively in biochemistry applications. It is a metalloenzyme with many isoforms, of which the most studied type is C. It catalyzes the oxidation of various or ...
(HRP), or NADH, NADPH using enzymes).
7-ethoxyresorufin, a compound used as the substrate in the measurement of
cytochrome P450
Cytochromes P450 (P450s or CYPs) are a Protein superfamily, superfamily of enzymes containing heme as a cofactor (biochemistry), cofactor that mostly, but not exclusively, function as monooxygenases. However, they are not omnipresent; for examp ...
(
CYP1A1
Cytochrome P450, family 1, subfamily A, polypeptide 1 is a protein that in humans is encoded by the ''CYP1A1'' gene. The protein is a member of the cytochrome P450 superfamily of enzymes.
Function
Metabolism of xenobiotics and drugs
CYP1A1 is ...
) 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(9''H'')-one (DDAO dye), a fluorescent dye used for oligonucleotide labeling.
References
{{reflist
Cell culture reagents
Redox indicators
Oxazine dyes
Amine oxides
Phenoxazines