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alamarBlue® Cell Viability Assay Protocol

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Introduction Learn more about alamarBlue®

Materials
Ordering Inf ormation
Protocol
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Introduction

Cell health can be monitored by numerous methods. Plasma membrane integrity, DNA synthesis, DNA content, enzyme activity, presence of ATP, and
cellular reducing conditions are know n indicators of cell viability and cell death. alamarBlue® cell viability reagent functions as a cell health indicator
by using the reducing pow er of living cells to quantitatively measure the prolif eration of various human and animal cell lines, bacteria, plant, and f ungi
allow ing you to establish relative cytotoxicity of agents w ithin various chemical classes. When cells are alive they maintain a reducing environment
w ithin the cytosol of the cell. Resazurin, the active ingredient of alamarBlue® reagent, is a non-toxic, cell permeable compound that is blue in color
and virtually non-fluorescent. Upon entering cells, resazurin is reduced to resoruf in, a compound that is red in color and highly f luorescent. Viable
cells continuously convert resazurin to resoruf in, increasing the overall fluorescence and color of the media surrounding cells.

alamarBlue® cell viability reagent is used to assess cell viability by simply adding the 10X, ready-to-use solution to mammalian or bacterial cells in
culture media (Figure 1). There is no requirement to aspirate media f rom cells or place cells in minimal media. Consequently, alamarBlue® reagent can
easily be used in a single tube or microtiter plate format in a “now ash” fashion. Simply add alamarBlue® reagent as 10% of the sample volume (i.e.,
add 10 μL alamarBlue® reagent to 100 μL sample), follow ed by a 1–4 hours incubation at 37ºC. Longer incubation times may be used f or greater
sensitivity w ithout compromising cell health (Figure 3 and see Frequently Asked Questions). The resulting f luorescence is read on a plate reader or
f luorescence spectrophotometer. Alternatively, the absorbance of alamarBlue® reagent can be read on a spectrophotometer. Finally, results are
analyzed by plotting fluorescence intensity (or absorbance) versus compound concentration.

Figure 1: alamarBlue® cell viability assay protocol. A 96-w ell plate containing the cells and the compounds to be tested is prepared using
standard methods. alamarBlue® reagent is added directly to each w ell, the plates are incubated at 37°C to allow cells to convert resazurin to
resoruf in, and the f luorescence (or absorbance) signal is measured. Results are evaluated by plotting the fluorescent (or absorbance) signal versus
compound concentration. This depicts an assay carried out in a 96-w ell plate, but the procedure is readily adaptable to other f ormats as w ell
(including 384-w ell plates and tubes of various volumes). If tubes are used, the sample is transferred to a cuvette prior to spectrophotometric
analysis.

alamarBlue® reagent can detect as f ew as 50 cells per w ell in a 96-w ell plate. To evaluate the sensitivity of alamarBlue® reagent, a serial dilution of
HUVEC cells w as perf ormed in a black, clear-bottom 96-w ell plate. Cells w ere then treated w ith alamarBlue® reagent and the fluorescence w as
measured 40 minutes and 18 hours later. Af ter 40 minutes, the fluorescence intensity of alamarBlue® reagent w as directly proportional to cell
number in the range of 500–50,000 cells. Af ter 18 hours, the fluorescence intensity of alamarBlue® reagent w as directly proportional to cell number
in the range of 50–5,000 cells, leading to more sensitive detection (Figure 3).

Figure 2A: Linearity of alamarBlue®. The f luorescence readout using alamarBlue®

w as linear over the range from ~500 to 50,000 cells af ter a 40 minute incubation.

Figure 2B: Sensitivity of alam arBlue®. This graph of the same 96-w ell plate show s
that the limit of detection of alamarBlue® can reach 50 cells per w ell w ith linearity to
5,000 cells per w ell by using an 18 h incubation. The horizontal line at ~450 RFU
represents the background fluorescence, calculated as three times the standard
deviation of the “no cell” control.

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Materials

M aterials required but not provided

Mammalian or bacterial cells in appropriate medium
Appropriate 96- or 384-w ell plates
Optional: 3% SDS in phosphate buffered saline (PBS), pH 7.4

Preparing Cells
Mam malian Cells—Adherent: Plate mammalian cells in a cell culture flask or dish, and allow cells to adhere and grow for approximately 4–
24 hours at 37°C and 5% CO2 bef ore proceeding w ith the assay.
Mam malian Cells—Suspension: Plate mammalian cells in a cell culture flask or dish, and use cells immediately for the assay or allow cells
to grow for up to 24 hours at 37°C and 5% CO2 before proceeding w ith the assay.
Bacterial Cells: For details, see ref erences 2 and 3. Notes alamarBlue® reagent is stable to multiple f reeze/thaw cycles and its activity is not
aff ected if the reagent is f rozen.

General Guidelines
Cell types assayed w ith alamarBlue® reagent include mammalian, bacterial2 (including biofilms 3), plant,4 and f ish cells.5 More specif ically
alamarBlue® reagent has been tested on hepatocytes, such as HepG2 cells, as w ell as cells of primary origin.5
Be sure to include appropriate assay controls. To minimize experimental errors, w e recommend making measurements f rom a minimum of 4–8
replicates of experimental and no-cell control samples.
You may need to determine the plating density and incubation time f or the alamarBlue® assay f or each cell type and use conditions such that
the assay is in the linear range.
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 w ill yield erroneous results as microbial contaminants also reduce alamarBlue® 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. Other media components, such as phenol red do not interf ere w ith the assay.
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Ordering Information

Sku Product Catalog Size Price Quantity

DAL1025 alamarBlue® 25 ml 164.00 USD

DAL1100 alamarBlue® 100 ml 354.00 USD

Protocol

Optional: Treat cells w ith the test compound 24–72 hours prior to performing the alamarBlue® cytotoxicity assay.

1. Add 1/10th volume of alamarBlue® reagent directly to cells in culture medium as described in Table 1.

Format Volum e of cells + m edium Volum e of 10x alam arBlue to add

Cuvette 1 mL 100 µL

96-w ell plate 100 µL 10 µL

384-w ell plate 40 µL 4 µL

2. Incubate for 1 to 4 hours at 37°C in a cell culture incubator, protected from direct light.

Note: Sensitivity of detection increases w ith longer incubation times. For samples w ith few er cells, use longer incubation times of up to 24
hours.

3. Record results using f luorescence or absorbance as follow s:

Fluorescence: Read f luorescence using a f luorescence excitation w avelength of 540–570 nm (peak excitation is 570 nm). Read
fluorescence emission at 580–610 nm (peak emission is 585 nm).

Absorbance: Monitor the absorbance of alamarBlue® at 570 nm, using 600 nm as a reference w avelength (normalized to the 600 nm
value).

Note: Fluorescence mode measurements are more sensitive. When fluorescence instrumentation is unavailable, monitor the absorbance of
alamarBlue® reagent. Assay plates or tubes can be w rapped in f oil, stored at 4°C, and read w ithin 1–3 days w ithout affecting the
fluorescence or absorbance values.

4. Optional: Add 50 μL 3% SDS directly to 100 μL of cells in alamarBlue® reagent to stop the reaction.
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FAQ's

General Questions

Q: How does alamarBlue® w ork?

A: Healthy living cells maintain a reducing state w ithin their cytosol. This “reducing potential” of cells converts alamarBlue® reagent into a detectable
f luorescent (or absorbent) product.

Q: Is alamarBlue® reagent toxic?

A: No. alamarBlue® reagent is a safe, non-toxic reagent to both the sample and user.

Q: Does alamarBlue® reagent need reconstitution?

A: No, alamarBlue® reagent is supplied as a 10X, ready-to-use solution.

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Protocol
Q: Can I use alamarBlue® reagent w ith suspension cells too?
http://www.foxitsoftware.com For evaluation only.
A: Yes. alamarBlue® reagent w orks on adherent and suspension mammalian cells.

Q: Can I use alamarBlue® reagent w ith non-mammalian cells, such as bacteria?

A: Yes, alamarBlue® reagent has been show n to w ork w ith bacterial2 and plant cells.4

Q: alamarBlue® reagent is not the most expensive cytotoxicity indicator on the market, does that mean it doesn’t w ork as w ell as other reagents?
A: Actually, alamarBlue® reagent is comparable to other often more expensive cytotoxicity indicators.

Q: Since alamarBlue® is an absorbance or f luorescence readout, is it as sensitive as a luminescence product?

A: alamarBlue® reagent is sensitive enough to detect less than 50 mammalian cells in a single w ell of a 96-w ell plate.

Storage Questions

Q: What if I left the alamarBlue® stock reagent at room temperature, overnight?

A: The reagent is stable f or up to 12 months w hen stored at room temperature (~22ºC).

Q: I accidentally froze the alamarBlue® stock reagent, can I still use it?
A: Yes. alamarBlue® reagent is stable to multiple f reeze/thaw cycles. Be sure to heat the reagent in a 37°C w ater bath and mix the reagent to ensure
a homogenous solution bef ore use.

Q: Do I need to protect alamarBlue® reagent from light?

A: Yes, alamarBlue® reagent is very slow ly converted into a fluorescent product over time, w hen exposed to light, thus leading to high background
values. Store the reagent, protected from light.

M ethods Questions

Q: What is the optimal incubation time and temperature of cells w ith alamarBlue® reagent?
A: Incubate the cells w ith alamarBlue® reagent for 1–4 hours at 37°C. For more sensitive detection w ith low cell numbers, increase the incubation
time for up to 24 hours.

Q: Can you incubate cells w ith alamarBlue® reagent overnight?

A: Yes. How ever, signals from higher cell density samples may have “saturated,” w hich means the linearity of reagent may have reached a plateau.
If this occurs, decrease the incubation time.

Q: What if I don’t have an instrument suitable f or reading f luorescence?

A: The absorbance of alamarBlue® reagent also changes depending on cell viability and prolif eration. Therefore, simply monitor the absorbance of
the reagent at 570 nm, w hile using 600 nm as a reference w avelength.

Q: Is alamarBlue® assay strictly an endpoint assay?

A: No. While alamarBlue® can be used as a terminal readout of a population of cells, the reagent can also be used to continuously monitor cell viability
and prolif eration in real time. Since alamarBlue® reagent is non-toxic, you can incubate cells w ith reagent and monitor f luorescence (or absorbance)
over time on the same sample.

Troubleshooting Questions

Q: What is the problem f or observing high background f luorescence values?

A: The reagent may be breaking dow n 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.

Q: Why are the f luorescence values so low in intensity?

A: Try increasing the incubation time of cells w ith alamarBlue® reagent, changing the instrument’s “gain” setting, and checking the instrument
f ilter/w avelength settings. Make sure to have positive controls (living cells) in the experimental design f or troubleshooting.

Q: Why are the f luorescence values so high that they are beyond the linear range of the instrument?
A: Try decreasing the incubation time or reducing the number of cells used in the experiment.
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References

1. Invest Ophthalmol Vis Sci 38, 1929 (1997)

2. Infect Immun 65, 3193 (1997)

3. J Antimicrob Chemother 57, 1100 (2006)

4. Phytochem Anal 12, 340 (2001)

5. Anal Biochem 344, 76 (2005)

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MP 01025 16–July–2008

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alamarBlue® Cell Viability Assay by Foxit PDF Creator © Foxit Software
Protocol
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