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Invitrogen™ alamarBlue™ and alamarBlue™ HS Cell Viability Reagents

Catalog No. DAL1025
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100 mL
Purity or Quality Grade:
HS Purified
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Catalog No. Quantity Purity or Quality Grade
DAL1025 25 mL Standard
DAL1100 100 mL Standard
A50100 25 mL HS Purified
A50101 100 mL HS Purified
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Catalog No. DAL1025 Supplier Invitrogen™ Supplier No. DAL1025
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Description

The alamarBlue cell viability reagents are ready-to-use resazurin-based solutions that function as cell health indicators by using the reducing power of living cells to quantitatively measure viability.

alamarBlue and alamarBlue HS cell viability reagents are ready-to-use, non-toxic, solutions based on resazurin. These reagents function as cell health indicators, enabling quantitative measurement of cell viability and proliferation using absorbance- or fluorescence-based microplate readers. alamarBlue uses the natural reducing power of living cells to convert resazurin to fluorescent resorufin. alamarBlue HS (High Sensitivity) Cell Viability Reagent is an improved version of alamarBlue that retains all the key alamarBlue reagent characteristics, but also provides increased sensitivity.

Features of the alamarBlue cell viability reagents include:

  • Robust and reliable performance—results in a large, highly reproducible dynamic range
  • Highly sensitive reagent with a linear response—detects as few as 50 cells per well for alamarBlue and as few as 20 cells per well for alamarBlue HS
  • Convenient add-and-read format—no mixing, no washing, no cell lysis
  • Compatible with either fluorescence- or absorbance-based microplate readers and instrumentation
  • Measures viability from many diverse cell types—including mammalian cells, bacteria, plants, and fungi
  • Provides a non-destructive assay platform, allowing for continuous monitoring of cell viability and proliferation over extended periods

Measuring changes in cell viability is a fundamental method for assessing cell health, determining genotoxicity, and evaluating anti-cancer drugs. Cell health can be monitored by detecting changes in several key indicators, including changes to plasma membrane integrity, DNA synthesis, DNA content, enzyme activity, presence of ATP, and cellular reducing environment. alamarBlue cell viability reagents are indigo-colored, non-toxic reagents that detect metabolically active cells and are used for the quantitative analysis of cell viability and proliferation. These alamarBlue reagents have broad applicability and can be used with various human and animal cell lines, bacteria, plants, and fungi. They are recommended for extended viability studies or when using a high cell density.

Mechanism of action

When added to cells, the alamarBlue cell viability reagents are modified by the reducing environment of viable cells and turn red in color and become highly fluorescent. This color change and increased fluorescence can be detected using absorbance (detected at 570 and 600 nm) or fluorescence (using an excitation between 530–560 and an emission at 590 nm).

Easy and reliable viability assay

To assay for viability, simply add the pre-mixed alamarBlue reagent to cells in complete medium (no wash or cell lysis steps required), incubate for 1–4 hours, and read using either an absorbance- or fluorescence-based plate reader. If necessary, longer incubation times may be used for greater sensitivity without compromising cell health. Since no lysis is required, the diluted alamarBlue solution can be removed and replaced with complete growth medium for further culturing of the cells. The assay can be performed using Thermo Scientific microplate readers such as Multiscan FC, Multiscan SkyHigh, Varioskan ALF, and Varioskan LUX, offering versatility for diverse research needs such as evaluation of compound effects, cytotoxicity, and cell proliferation in various experimental settings.

alamarBlue HS—a highly purified version of alamarBlue with improved performance

High background fluorescence caused by various contaminants reduces the performance of resazurin-based reagents. To improve the performance, an innovative purification process was developed that removes the contaminating resorufin, resulting in the highly pure resazurin used in the alamarBlue HS Cell Viability Reagent. alamarBlue HS has improved performance, which results in a >50% decrease in background fluorescence and a >100% increase in the signal-to-background ratio. Furthermore, the alamarBlue HS reagent displays an extended viability detection time window for a wide variety of organisms and thus can be used with various human and animal cell lines, bacteria, plants, and fungi, resulting in quantitative measurement of cell viability and proliferation.

Benefits of the alamarBlue HS Cell Viability Reagent over alamarBlue include:

  • Removal of contaminants from resazurin—displays a >50% reduction in background fluorescence
  • Signal-to-background ratio increased by >100%—results in large assay signal window
  • Highly sensitive reagent with a linear response—detects as few as 20 cells per well

alamarBlue HS reagent provides a high-sensitivity, non-toxic, and versatile solution for measuring cell viability and proliferation across various research applications. Its enhanced sensitivity makes it particularly valuable in contexts where detecting small changes in cell activity is essential.

High-throughput compatibility and automation potential

The homogeneous, add-and-read format of the alamarBlue cell viability reagents makes them exceptionally well-suited for high-throughput screening applications and automation platforms. The simplicity of the protocol, combined with the elimination of wash and lysis steps, facilitates rapid and efficient processing of large sample sets. This feature is particularly valuable for drug discovery, toxicity screening, and other applications requiring the analysis of multiple compounds or conditions simultaneously.

Evaluate cell proliferation and viability in diverse applications

alamarBlue cell viability reagents are widely used in diverse research applications. They are ideal for drug screening and cytotoxicity testing, allowing researchers to assess the effects of potential drug candidates on cell viability. In cancer research, alamarBlue is used to evaluate the efficacy of anti-cancer compounds and monitor cell proliferation. alamarBlue reagents are also employed in environmental testing to assess the toxicity of chemicals and pollutants, as well as in microbial viability studies for antibiotic testing.

Specifications

Cell Type Bacteria, Eukaryotic Cells, Fungal Cells
Color Blue
Concentration 10X
Content And Storage Store in refrigerator at 2°C to 8°C
Protect from light.
Description alamarBlue™ Cell Viability Reagent
Detection Method Colorimetric, Fluorescence
For Use With (Application) Viability Assay
For Use With (Equipment) Spectrophotometer, Microplate Reader
Product Type Cell Viability Reagent
Dye Type Resazurin
Emission 590
Excitation Wavelength Range 530-560 nm
Form Liquid
Format 96-well plate, Cuvettes, 384-well plate
Incubation Time 1 to 4 hr. (Up to 24 hr. for low cell numbers)
Product Line alamarBlue
Purity or Quality Grade Standard
Quantity 25 mL
Shipping Condition Wet Ice
Throughput High-throughput Compatible
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Frequently Asked Questions (FAQs)

MTT cell proliferation plate assays require cellular metabolism to modify the reagent and thus, only live cells will be counted. Is this true for CyQUANT Direct Cell Proliferation Assay, too?

This is true for MTT (as well as XTT, AlamarBlue Cell Viability Reagent, and PrestoBlue Cell Viability Reagent). CyQUANT Direct will also only count live cells, but for a different reason. The dye in it is a green-fluorescent nucleic acid stain, which will bind to DNA in all cells, live or dead, without the need for cellular metabolism. However, there is a non-cell-permeable quenching reagent in the kit which will both quench extracellular fluorescence (and thus this is a no-wash assay) AND will quench the fluorescence in dead cells (but not live cells).
MTT cell proliferation assays require cellular metabolism to turn over the reagent, and thus only live cells will be counted. Is this true for CyQUANT Direct as well?

This is true for MTT (as well as XTT, AlamarBlue Cell Viability Reagent, and PrestoBlue Cell Viability Reagent). CyQUANT Direct will also only count live cells, but for a different reason. The dye is a green-fluorescent nucleic acid stain, which will bind to DNA in all cells, live or dead, without the need for cellular metabolism. However, there is a cell impermeant quenching reagent in the kit which will quench both extracellular fluorescence (and thus this is a no-wash assay) and the fluorescence in dead cells (but not live cells).
Why would I use alamarBlue HS Cell Viability Reagent instead of the original alamarBlue Cell Viability Reagent?

alamarBlue HS Cell Viability Reagent provides a lower background, suitable for analyzing a lower number of cells per well. Also, the removal of impurities in the 'HS' reagent limits any artifacts that may be critical to your cells' viability.
Does alamarBlue HS Cell Viability Reagent have the same properties as the original alamarBlue Cell Viability Reagent?

Yes and in addition, alamarBlue HS Cell Viability Reagent has a higher purity which results in a lower background.

What is the difference between the original alamarBlue Cell Viability Reagent (Cat. Nos. DAL1025, DAL1100) and alamarBlue HS Cell Viability Reagent (Cat. Nos. A50100, A50101)?

Both products utilize resazurin as the indicator dye. alamarBlue HS Cell Viability Reagent undergoes a purification process that removes any residual resorufin that may be present from the standard manufacturing process for resazurin. This purification results in a product that has very low background. alamarBlue HS Cell Viability Reagent may be used on samples with as few as 20 cells/well, compared to a minimum of 50 cells/well for the original alamarBlue Cell Viability Reagent.
Note: Minimum cell number/well is also dependent upon reducing activity of the cell type. Results may vary.

With alamarBlue reagent, my readings across the plate are erratic. What has happened?

One reason could be that the dye in your alamarBlue reagent has precipitated, resulting in varying dye concentration. In such cases, the reagent should be warmed to 37 degrees C and shaken or swirled to ensure that all components are completely in solution. Another cause can be pipetting issues. Ensure that your pipettor has been calibrated and the pipette tips are securely anchored prior to pipetting.

I am using alamarBlue reagent. Why are the fluorescence values so high that they are beyond the linear range of the instrument?

Decrease the incubation time and/or reduce the number of cells used in the experiment.

I am using alamarBlue reagent and my fluorescence values are very low. What went wrong?

We recommend increasing the incubation time of cells with alamarBlue reagent, changing the instrument's gain or voltage setting, and checking the instrument filter/wavelength settings. Make sure to have positive controls (untreated, living cells) in the experimental design as a control.

I am using alamarBlue reagent and am observing high background fluorescence. What is the problem?

The reagent may be breaking down due to exposure to light. Be sure to store alamarBlue reagent in the dark and do not expose the reagent to direct light for long periods of time.
It looks like my alamarBlue dye has precipitated. What should I do?

Because the indicator is a multicomponent solution, we recommend that frozen alamarBlue reagent be warmed to 37 degrees C and shaken or swirled to ensure that all components are completely in solution.

I left the alamarBlue stock reagent at room temperature over the weekend. Is it still good to use?

The reagent is stable for up to 12 months when stored at room temperature (approximately 22 degrees C).

I accidentally froze the alamarBlue stock reagent, can I still use it?

Yes. alamarBlue reagent is stable to multiple freeze/thaw cycles. Be sure to warm the reagent in a 37 degrees C water bath and mix it well to ensure a homogenous solution before use.

How do alamarBlue reagent and PrestoBlue reagent differ from resazurin and C12-resazurin?

alamarBlue reagent and PrestoBlue reagent contain resazurin in a proprietary stabilizing formulation that allows for a convenient “mix, incubate, and read” protocol. PrestoBlue reagent is an improvement in the formulation of alamarBlue reagent that allows for much faster staining (typically 10 minutes vs. 1-4 hours to obtain a similar signal and sensitivity). C12-resazurin is a derivative of resazurin that has better cellular retention and thus allows for analysis on a flow cytometer and multiplexing with viability indicators and other biomarkers.
Which cell proliferation assays can I use with 3D culture systems?

alamarBlue Cell Viability Reagent, PrestoBlue Cell Viability Reagent, and CyQUANT Cell Proliferation Assay Kit (Cat. No. C7026) have been validated for use with the AlgiMatrix 3D Culture System and should also work with other 3D culture systems. For further details, please refer to this poster (https://aacrjournals.org/cancerres/article/70/8_Supplement/3234/563757/Abstract-3234-AlgiMatrixT-3-D-cell-culture-system) and protocol (http://www.thermofisher.com/us/en/home/references/protocols/cell-culture/3-d-cell-culture-protocol/algimatrix-viability-using-cyquant.html).

I wish to assay the viability of my cells over several days. Can I use any of your LIVE/DEAD imaging kits to do this?

No, it is not possible to assay the viability of the same population of cells longer than a few hours; you will need to use replicate samples. DNA binding dyes are toxic to cells; stained cells should be imaged as soon as possible after staining. Calcein AM is not retained in cells and may be actively effluxed out in the range from minutes to several hours, dependent upon the cell type. Calcein AM is not toxic to cells, so it can be added repeatedly to the same samples. You can assay the proliferation of the same sample of cells over several days using alamarBlue reagent or PrestoBlue reagent, as these dyes are non-toxic to cells.
How does media affect the alamarBlue Cell Viability Reagent?

Fetal bovine serum (FBS) and bovine serum albumin (BSA) cause some quenching of fluorescence. We recommend using the same serum concentration in controls to account for this quenching. Assuming the media does not contain reducing agents, other media components do not interfere with the assay. There is no interference from the presence of phenol red in the growth medium if the pH has not changed significantly. At or near neutral pH, the presence of phenol red merely shifts the values approximately 0.03 units higher. Ideally, the assay should be done in phenol red-free medium.

How do I determine the optimal plating density and optimal incubation time for the alamarBlue reagent assay?

You may need to determine the plating density and incubation time for the alamarBlue assay for each cell type and use conditions such that the assay is in the linear range.

Plating Density:
alamarBlue reagent measures cell proliferation most accurately when the cells are in the exponential growth phase. If the cell density is too high, cell proliferation will decrease, giving less reduction of the alamarBlue reagent than would have been expected. At low cell density, the slower growth rate could result in insignificant alamarBlue reduction. A cell density of 1 x 10E4 cells/mL is generally recommended for animal cells, with cells in the exponential phase. However, since cells vary in their proliferation rate, it is impossible to recommend a cell density which is suitable for all experiments. Instead we recommend performing a control experiment to determine the optimum cell density for your studies. A detailed protocol is provided in the product manual.

Incubation Time:
It is equally important to optimize the incubation time for the alamarBlue reagent. Incubate the cells with alamarBlue reagent for 1-4 hours at 37 degrees C or, at an optimal temperature for the species/cell type. For more sensitive detection with low cell numbers, increase the incubation time for up to 24 hours. If you plan to use longer incubation time (overnight), be sure to maintain sterile conditions during reagent addition and incubation to avoid microbial contaminants. Contaminated cultures will yield erroneous results as microbial contaminants also reduce alamarBlue reagent. A detailed protocol is provided in the product manual.

alamarBlue reagent contains proprietary buffering agents which maintain the stability and solubility of both the oxidized and reduced forms of the alamarBlue reagent and other components in the solution. Over extended incubation times with cells, the buffering capacity may be altered to such an extent that reagent solubility and stability may be affected, resulting in a loss of signal.

Alternatively, if the length of the experiment is longer than the optimal alamarBlue incubation time, we suggest that an endpoint test is used. This type of experiment is particularly useful for cell proliferation studies over days, weeks and months. alamarBlue reagent can be used for long-term cell proliferation studies where measurements are taken repeatedly. For these types of studies, we recommend that aliquots of the cell medium/ suspension are taken at each time point during incubation with alamarBlue reagent prior to an endpoint.

What kind of controls should I use with the alamarBlue Cell Viability Reagent?

We recommend including appropriate assay controls. To minimize experimental errors, we recommend making measurements from a minimum of 4-8 replicates of experimental and no-cell control samples. Some suggestions are provided below:

No-cell controls: Wells that contain only culture media so that the background fluorescence can be determined and subtracted from experimental wells. We recommend using the same serum concentration in controls. Phenol red may interfere with the assay; ideally the assay should be performed using Phenol red-free media.
No-cell + experimental compound controls: Wells that contain only cell culture media and added compound to assess potential background fluorescence or absorbance caused by the drug/chemical used in the experiment.
Untreated-cell controls: If you're treating cells with a compound, we recommend that you plate wells of untreated cells.
Solvent controls: If you're treating cells with a compound dissolved in a solvent such a DMSO, ethanol, etc., we recommend that you plate wells of cells with added solvent only (same volume added to experimental samples, no compound); this control should provide similar results as untreated cell controls. If results from these samples are different from untreated cell controls, the sample may be sensitive to the solvent.

I am planning to use the alamarBlue Cell Viability Reagent. How do I measure results with fluorescence and absorbance?

Fluorescence: Read fluorescence using an excitation wavelength between 530-570 nm (peak excitation is 570 nm). Read fluorescence emission between 580-610 nm (peak emission is 585 nm).
Absorbance: Monitor the absorbance of alamarBlue reagent at 570 nm, using 600 nm as a reference wavelength (normalized to the 600 nm value).
Note: Fluorescence detection is more sensitive. When fluorescence instrumentation is unavailable, monitor the absorbance of alamarBlue reagent. With most animal cells, assay plates or tubes can be wrapped in foil or plastic wrap (to prevent evaporation), stored at 4 degrees C, and read within 1-3 days without affecting the fluorescence or absorbance values. This may not hold true for bacterial, algal, protozoan, or fungal samples.

What are the storage conditions for alamarBlue Cell Viability Reagent?

alamarBlue Cell Viability Reagent compound is light sensitive and should be stored in the dark. The product may be stored for 12 months at room temperature. The expiration date is given on the product label. If shelf life beyond 12 months is desired, storing at 2-8 degrees C increases shelf life to 20 months. alamarBlue reagent may also be frozen at greater than -70 degrees C indefinitely. Because the indicator is a multicomponent solution, we recommend that frozen alamarBlue reagent be warmed to 37 degrees C and shaken to ensure all components are completely in solution.
What cell types can be assayed with alamarBlue reagent?

alamarBlue reagent has been used with many different cell species: mammalian, avian, amphibian, fish, diatoms, bacteria, plant cells, fungi and others-and cell samples, such as immortalized cell cultures, primary cell cultures, cancer cells, stem cells, etc. Also, alamarBlue reagent has been used to measure viability of tissue samples such as heart valves and cells cultured in 3D matrices such as AlgiMatrix matrix. There are many references available for this application. You can find them by searching for alamarBlue and your cell type of interest. For example, using Google Scholar, use the search term ‘alamarBlue and cancer cells' or ‘alamarBlue and tissue'.
Is alamarBlue reagent toxic?

While most reports in the literature suggest that solutions containing resazurin, the active ingredient in alamarBlue reagent, are not toxic to cells, other reports clearly show that cell viability is affected depending on the length of exposure and concentration of resazurin to which they are subjected.

How does the alamarBlue Cell Viability Reagent work?

alamarBlue Cell Viability Reagent is a proven cell viability and proliferation indicator. It contains the active ingredient, resazurin, a non-toxic, cell permeable, non-fluorescent blue indicator dye. Healthy living cells maintain a reducing state within their cytosol. Upon diffusion into cells, the natural reducing potential of living cells converts resazurin to the very bright red, cell permeable fluorescent dye, resorufin. Resazurin is reduced by the removal of oxygen.

Viable cells continuously convert resazurin to resorufin, thereby generating a quantitative measure of viability, proliferation, and cytotoxicity. The net result is measurable change in the color and fluorescence intensity. The amount of fluorescence or absorbance is proportional to the number of living cells and corresponds to the cells' metabolic activity. Damaged and nonviable cells have lower metabolic activity and thus generate a proportionally lower signal than healthy cells.
Do you offer any products to measure neuronal cell health?

PrestoBlue Cell Viability Stain and CyQUANT Cell Proliferation Assay Kit can be used. We also offer a Neurite Outgrowth Staining Kit (Cat. No. A15001). More information about our different assays for neuronal cell health can be found here (https://www.thermofisher.com/us/en/home/life-science/cell-analysis/neuroscience/neuronal-cell-health-assays.html).
Can other cell viability reagents be used with AlgiMatrix Matrix?

Other than the Invitrogen alamarBlue Cell Viability Reagent, AlgiMatrix Matrix is also compatible with Invitrogen PrestoBlue Cell Viability Reagent and Invitrogen CyQuant Direct Cell Proliferation Assay. The protocol is almost the same as the 2D cell culture, but it needs the blank AlgiMatrix Matrix as a blank control.

What is the function of the Invitrogen alamarBlue reagent assay?

The Invitrogen alamarBlue reagent assay is a useful biological assay that monitors the health and function of cells by monitoring their reducing functions.

For Research Use Only. Not for use in diagnostic procedures.