non-fluorescent dye; redox indicator

Recommended protocol (This is our suggested protocol, which should be adjusted to suit your particular needs as it simply offers guidance).
Cell Viability:
1. The resazurin solution should be thawed in a 37°C water bath.
2. After adding cells to a 96-well plate, wash them with PBS (protect from light).
3. After removing the PBS wash solution, add 500 μL of resazurin solution (1μg/mL).
4. Incubate the plate for 30 minutes (the length of the incubation period varies depending on the type and quantity of cells).
5. Resorufan fluorescence can be measured using a microplate reader (Ex=530-560 nm, Em=590 nm).

Assay Chemistry and Redox Principle [2]
Alamar Blue monitors the reducing environment of the living cell. The active ingredient is resazurin (IUPAC name: 7-hydroxy-10-oxidophenoxazin-10-ium-3-one), also known as diazo-resorcinol, azoresorcin, resazoin, resazurin, which is water-soluble, stable in culture medium, is non-toxic and permeable through cell membranes. Continuous monitoring of cells in culture is therefore permitted. Resazurin was first used to assess bacterial or yeast contamination in milk by Pesch and Simmert in 1929. It is a blue non-fluorescent dye that is reduced to the pink-colored, highly fluorescent resorufin. Resazurin solution is highly dichromatic based on Kreft's dichromaticity index (DI). The dye acts as an intermediate electron acceptor in the electron transport chain without interference of the normal transfer of electrons. The oxidation-reduction potential of Alamar Blue is +380 mV at pH 7.0, 25 °C. Alamar Blue, therefore, can be reduced by NADPH (Eo = 320 mV), FADH (Eo = 220 mV), FMNH (Eo = 210 mV), NADH (Eo = 320 mV), as well as the cytochromes (Eo = 290 mV to +80 mV). As the indicator dye accepts electrons, it changes from the oxidized, non-fluorescent, blue state to the reduced, fluorescent, pink state. In addition to mitochondrial reductases, other enzymes (such as the diaphorases (EC 1.8.1.4, dihydrolipoamine dehydrogenase), NAD(P)H:quinone oxidoreductase (EC 1.6.99.2) and flavin reductase (EC 1.6.99.1) located in the cytoplasm and the mitochondria may be able to reduce Alamar Blue. Hence, Alamar Blue reduction may signify an impairment of cellular metabolism and is not necessarily specific to interruption of electron transport and mitochondrial dysfunction. This change from oxidized to reduced state allows flexibility of detection where measurements can be quantitative as colorimetric and/or fluorometric readings (the latter being more sensitive) or qualitative as a visible change in color indicating presence or absence of viable cells. Spectrophotometric absorbance is taken at two wavelengths (570 and 600 nm or 540 and 630 nm, although other filters can be used with a correction factor to be incorporated in calculations). Absorbance values will vary according to microtiter plate (flat-bottomed or rounded and manufacturer). Fluorescence signals are measured at an excitation wavelength at 530–560 nm and an emission wavelength at 590 nm.

The identification of rapid, reliable, and highly reproducible biological assays that can be standardized and routinely used in preclinical tests constitutes a promising approach to reducing drug discovery costs and time. This unit details a tandem, rapid, and reliable cell viability method for preliminary screening of chemical compounds. This assay measures metabolic activity and cell mass in the same cell sample using a dual Resazurin/sulforhodamine B assay, eliminating the variation associated with cell seeding and excessive manipulations in assays that test different cell samples across plates. The procedure also reduces the amount of cells, test compound, and reagents required, as well as the time expended in conventional tests, thus resulting in a more confident prediction of toxic thresholds for the tested compounds. [1]

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Accurate prediction of the adverse effects of test compounds on living systems, detection of toxic thresholds, and expansion of experimental data sets to include multiple toxicity end-point analysis are required for any robust screening regime. Alamar Blue is an important redox indicator that is used to evaluate metabolic function and cellular health. The Alamar Blue bioassay has been utilized over the past 50 years to assess cell viability and cytotoxicity in a range of biological and environmental systems and in a number of cell types including bacteria, yeast, fungi, protozoa and cultured mammalian and piscine cells. It offers several advantages over other metabolic indicators and other cytotoxicity assays. However, as with any bioassay, suitability must be determined for each application and cell model. This review seeks to highlight many of the important considerations involved in assay use and design in addition to the potential pitfalls[2].

rat LDLo oral 500 mg/kg National Academy of Sciences, National Research Council, Chemical-Biological Coordination Center, Review., 5(24), 1953
mouse LDLo intravenous 179 mg/kg Toxicology and Applied Pharmacology., 44(225), 1978 [PMID:79242]

[1]. Determination of Metabolic Viability and Cell Mass Using a Tandem Resazurin/Sulforhodamine B Assay. Curr Protoc Toxicol. 2016 May 4;68:2.24.1-2.24.15.

[2]. Rampersad SN. Multiple applications of Alamar Blue as an indicator of metabolic function and cellular health in cell viability bioassays. Sensors (Basel). 2012;12(9):12347-60.

Resazurin is a phenoxazine. Used as a pH indicator, to detect hyposulfite, and in food research (reductase test).

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Indicators and Reagents
Substances used for the detection, identification, analysis, etc. of chemical, biological, or pathologic processes or conditions. Indicators are substances that change in physical appearance, e.g., color, at or approaching the endpoint of a chemical titration, e.g., on the passage between acidity and alkalinity. Reagents are substances used for the detection or determination of another substance by chemical or microscopical means, especially analysis. Types of reagents are precipitants, solvents, oxidizers, reducers, fluxes, and colorimetric reagents.

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Resazurin caused oxidative stress that lead to autophagy and cell death through cellular production of reactive oxygen species (ROS) and mitochondrial impairment in HL-60 and ‘Jurkat’ leukemia cells. The dye itself caused a reduction in leukemia cell proliferation which has important implications in toxicity and chemo-resistance screening. This emphasizes the importance of empirically testing positive and negative controls for the assay to ensure no non-specific interaction effects.

C12H7NO4

229.19

251.019

550-82-3

Resazurin sodium;62758-13-8

11077

Typically exists as solid at room temperature

1.57g/cm3

477.2ºC at 760mmHg

242.4ºC

1.74

2.47

1

4

0

17

463

0

C1=CC2=C(C=C1O)OC3=CC(=O)C=CC3=[N+]2[O-]

PLXBWHJQWKZRKG-UHFFFAOYSA-N

InChI=1S/C12H7NO4/c14-7-1-3-9-11(5-7)17-12-6-8(15)2-4-10(12)13(9)16/h1-6,14H

7-hydroxy-10-oxidophenoxazin-10-ium-3-one

Azoresorcin; Diazoresorcinol; Resazurin

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)