Chapter 5
Assessment of Cell Proliferation with Resazurin-Based
Fluorescent Dye
Ewa M. Czekanska
Abstract
The Alamar Blue assay is based on enzymatic reduction of indicator dye by viable cells and serves as an
effective tool for assessing cell proliferation and as a screening technique. It can be applied in studies
concentrating on animal, plant, yeast, and bacteria cells. Among the various methods for cell viability and
cytotoxicity, it utilises all features of ideal and reliable test; it is one-step, sensitive, safe, non-toxic for cells,
and cost-effective.
Key words: Cell proliferation, Resazurin, Alamar Blue, Metabolic activity, Oxidoreductases
1. Introduction
The resazurin-based assay was first introduced in the late 1920s
to investigate the sanitary condition of milk (1–3). Later, it was
applied to plant metabolism studies (4), for assessing semen qual-
ity (5) and antifungal susceptibility testing (6). Due to many
advantages of resazurin-based assay, it has also become a useful
tool for investigations of toxicants (7–9). Moreover, the simplic-
ity, safety, homogenous nature, and sensitivity give this assay a
predominant position over the other classic tests used for estimat-
ing cell viability and proliferation (10, 11).
There are many assays available in the market that are com-
posed of resazurin sodium salt dye to monitor in vitro mamma-
lian cell proliferation, such as Alamar Blue (Alamar Biosystems;
Invitrogen; AbD Serotec; Arcus), CellTiter Blue (Promega) and
Cell toxicity Colorimetric/Fluorometric Assay (Sigma–Aldrich).
The resazurin is a blue weakly fluorescent indicator dye that
changes into highly fluorescent pink resorufin in response to
irreversible chemical reduction (Fig. 1).
Martin J. Stoddart (ed.), Mammalian Cell Viability: Methods and Protocols, Methods in Molecular Biology, vol. 740,
DOI 10.1007/978-1-61779-108-6_5, © Springer Science+Business Media, LLC 2011
27
28 Czekanska
Fig. 1. Mechanism of reduction of resazurin to resorufin.
Inside the cell, Alamar Blue undergoes enzymatic reduction
in mitochondria due to the activity of enzymes such as: flavin
mononucleotide dehydrogenase, flavin adenine dinucleotide
dehydrogenase, nicotinamide adenine dehydrogenase, and cyto-
chromes (12). It was also noted that cytosolic and microsomal
enzymes have abilities to reduce resazurin. Moreover, the extent
of mitochondrial reduction is similar to cytosolic reduction driven
by NADPH:quinine oxidoreductase, flavin reductase, and cyto-
chromes (13). The red resorufin is excreted outside the cells to
the medium which results in visible colour change from blue to
pink. Hence, the rate of reduction based on colour change, which
can be quantified colorimetrically or fluorometrically, reflects the
number of viable cells. Alamar Blue is very sensitive and, depend-
ing on cell type and incubation time, is linear in the range of
50–50,000 cells in 96-well plate.
The maximum absorbance for resazurin/resorufin is
605/573 nm, whereas the maximum peak of excitation/emission
spectra for resorufin is 579/584 nm. It is recommended to carry
out the measurements using fluorescence as it requires fewer cal-
culations and is more sensitive due to considerable overlap of the
absorbance spectra for oxidised and reduced forms of dye. To
monitor the reduction process of Alamar Blue dye by cells wide
range of fluorescence filters can be used: 530–570 nm for excita-
tion and 580–620 nm for emission.
2. Materials
1. Cell culture incubator.
2. Fluorescence plate reader with excitation 530–570 nm
and emission 580–620 nm filter pair.
3. Osteoblastic cell line MC3T3-E1 cells.
4. Cell culture medium (DMEM with 10% FBS and 1%
antibiotics).
5. Cell culture treated polystyrene 24-well plates.
Assessment of Cell Proliferation with Resazurin-Based Fluorescent Dye 29
6. 96-Well opaque-walled white plate.
7. 0.1 M phosphate-buffered saline (PBS) solution, pH 7.4.
8. Alamar Blue.
9. Light-shield tubes or 15-ml centrifuge tubes wrapped in alu-
minium foil.
3. Methods
The following protocol is optimised for 24-well tissue culture
plates with 1.9 cm2 surface area. Volumes of reagents are given
per well and they should be adjusted accordingly if other sizes are
used. The Alamar Blue assay can be optimised and applied to vari-
ous cell types (see Note 1), including primary cells obtained after
extraction from tissue and immortalised cell line. For the assay,
exponentially expanded cells should be plated after trypsinisation.
It is not recommended to use cells directly after thawing from
cryopreservation. As an example in this protocol MC3T3-E1 cells
cultured in DMEM with 10% FCS and 1% antibiotics will be used
(see Note 2).
3.1. Cell Plating 1. Seed cells in 24-well tissue culture plates with 500 ml of
for Standard Curve medium at the following densities: 1,000; 2,000; 5,000;
10,000; 20,000; 40,000; 80,000; 100,000. Include four
samples for each density.
2. Transfer plates to the incubator (37°C with an atmosphere of
5% CO2 and 95% humidity) and let them attach for 5 h. After
that time check if all cells are attached to the surface of the
well; if not incubate longer till all cells are attached.
3. Prepare 10% solution of Alamar Blue in culture medium in a
light-shield tube (see Notes 3 and 4). Calculate the amount
needed by multiplying the amount of samples by the volume
of solution per well (300 ml). In calculations include three
no-cell controls.
4. Carefully aspirate medium from each well and add 300 ml of
fresh medium containing a 10% solution of Alamar Blue to
the wells.
5. Wrap culture plates in aluminium foil and incubate for 4 h in
cell culture incubator at 37°C with an atmosphere of 5% CO2
and 95% humidity.
6. Transfer a 150-ml aliquot of each sample and media with dye
alone to a 96-well plate in duplicates.
7. Read fluorescence at excitation 530–570 nm and emission
580–620 nm wavelength on plate reader (see Note 5).
30 Czekanska
8. Calculate the results by averaging the values obtained for each
density and subtracting the average value of no-cell control.
Plot the results of fluorescence/cell number and define the
equation. For MC3T3-E1 cells the following equation was
obtained: f (x) = 0.0587x + 138.14.
3.2. Cell Plating 1. Seed 10,000 cells per well in 24-well tissue culture plates with
for Estimating 500 ml of medium (four samples for each time point; see Note 6).
Proliferation 2. At each time point, aspirate the media from four wells and wash
samples with 500 ml of 0.1 M PBS solution (see Note 7).
3. Add 300 ml of fresh medium containing a 10% solution of Alamar
Blue to the wells (see Note 8). Include three no-cell controls.
4. Proceed according to steps 5–7 in previous section.
5. Calculate the results by averaging fluorescent values of the
samples and subtracting average value of samples without
cells.
6. Calculate cell number at each time point using the equation
defined by standard curve.
4. Notes
1. Before performing the assay for the first time, optimise exper-
imental parameters, such as the incubation time, amount of
cells, and amount of Alamar Blue used. This is very important
as various cell types have unique metabolic capacity to reduce
resazurin to resorufin. Here, predetermined 4-h incubation
time and 10% solution of the dye were used for densities
1,000–100,000 cells/well. However, these conditions may
not be optimal for other cell types. Hence, perform the
screening assays with various cell densities, at least three con-
centrations of Alamar Blue and incubation times. Incubating
cells for too long with resazurin solution results in a second-
ary reduction of pink fluorescent resorufin into non-
fluorescent colourless hydroresorufin which further leads to
aberrant and inaccurate results. Table 1 illustrates the empiri-
cally determined requirements for various cell lines (14).
2. Resazurin can be reduced by commonly known antioxidants,
such as ascorbic acid, cysteine, dithionite, and dithiothreitol
(15). When using culture medium supplemented with ascorbic
acid the optimal incubation time may vary from culture in the
same type of medium without the ascorbic supplementation.
3. Depending on manufacturer Alamar Blue can be stored at −20°C,
4°C, or at room temperature. Generally, lower temperatures
increase the product stability. If −20°C storage conditions
Assessment of Cell Proliferation with Resazurin-Based Fluorescent Dye 31
Table 1
The recommended conditions for the Alamar Blue assay
in 96-well plate for normal and cancer cell lines. Reprinted
from (14) with permission from Elsevier
Optimal
Linear range (cell Optimal AB incubation
Cell line number × 104) concentration (%) time (h)
Normal cell lines
BALB/3T3 0.05–2 4 3
CHO-K1 0.05–3 10 3
NJTIDF 4054 0.05–2 4 3
Ovarian cancer cell lines
2008 0.05–3 4 1
IGROV-1 0.05–3 10 1
OVCAR-3 0.05–3 4 3
SK-OV-3 0.05–3 4 3
Leukaemia cell lines
HL-60 0.5–5 10 6
K-562 0.5–5 10 6
MOLT-4 0.5–5 10 6
are used, reduce the freeze–thaw cycles by 10. For this, the
best is to aliquot the stock solution.
4. Resazurin and resorufin are light-sensitive and need to be
protected from light otherwise it results in decreased
sensitivity.
5. It is recommended to read the plate on the day an experiment
is performed. If it is not possible, 96-well plate with sample
aliquots can be stored at 4°C wrapped in foil and measurement
can be taken within 1–3 days without affecting fluorescence
or absorbance. However, when measuring fluorescence, it has
to be kept in mind that temperature affects the fluorescence
values. Thus, to keep the conditions of fluorescence readings
constant wait till refrigerated plate is warmed up to the ambi-
ent temperature before reading.
6. Optionally, the reaction can be stopped and stabilised after
the incubation by adding 50 ml of 3% SDS in PBS (pH 7.4)
per 100 ml of original culture volume. Plates can be stored at
ambient temperature wrapped in foil up to 24 h.
7. Although Alamar Blue is known as a non-toxic in vitro assay
in short-time exposure, the prolonged contact of cells with
resazurin may affect the reduction rate and cell viability (16).
Thus, it is possible to perform more than one type of assay
32 Czekanska
on the same sample, but for proliferation assessment it is
better to use Alamar Blue as end point assay.
8. Alamar Blue should not be added directly to cultured cells in
long-time experiments as protein concentration may affect
the results by quenching the fluorescence signal (17). The
Alamar Blue solution at determined concentration should be
prepared freshly each time and before applying, samples
should be washed with PBS to reduce the risk of different
protein concentrations generated during the culturing time.
References
1. Twigg R.S. (1945) Oxidation-reduction 10. Larson E. M., Doughman D. J., Gregerson D.
aspects of resazurin. Nature, 155:401–402 S., and Obritsch W. F. (1997) A new, simple,
2. Nixon M. C. and Lamb A. B. (1945) Resazurin nonradioactive, nontoxic in vitro assay to
Test for Grading Raw Milk. Can J Comp Med monitor corneal endothelial cell viability. Invest
Vet Sci, 9(1):18–23 Ophthalmol Vis Sci, 38(10):1929–1933
3. Ali-Vehmas T., Louhi M., and Sandholm M. 11. Jonsson K. B., Frost A., Larsson R., Ljunghall
(1991) Automation of the resazurin reduc- S., and Ljunggren O. (1997) A new fluoro-
tion test using fluorometry of microtitration metric assay for determination of osteoblastic
trays. Zentralbl Veterinarmed B, 38(5): proliferation: effects of glucocorticoids and
358–372 insulin-like growth factor-I. Calcif Tissue Int,
4. De Jong D. W. and. Woodlief W. G. (1977) 60(1):30–36
Fluorimetric assay of tobacco leaf dehydroge- 12. O’Brien, J., Wilson, I., Orton, T., and Pognan,
nases with resazurin. Biochim Biophys Acta, F. (2000) Pognan. Investigation of the Alamar
484(2):249–259 blue (resazurin) fluorescent dye for the assess-
5. Glass R. H., Ericsson S. A., Ericsson R. J., ment of mammalian cell cytotoxicity. Eur. J.
Drouin M. T., Marcoux L. J., and Sullivan H. Biochem, 267, 5421–5426.
(1991) The resazurin reduction test provides 13. Gonzalez R. J. and Tarloff J. B. (2001)
an assessment of sperm activity. Fertil Steril, Evaluation of hepatic subcellular fractions for
56(4):743–746 Alamar blue and MTT reductase activity.
6. To W. K., Fothergill A. W., and Rinaldi M. G. Toxicol In Vitro, 15(3):257–259
(1995) Comparative evaluation of macrodilu- 14. Nakayama G. R., Caton M. C., Nova M. P.,
tion and alamar colorimetric microdilution and Parandoosh Z. (1997) Assessment of the
broth methods for antifungal susceptibility Alamar Blue assay for cellular growth and
testing of yeast isolates. J Clin Microbiol, viability in vitro. J Immunol Methods,
33(10):2660–2664 204(2):205–208
7. O’Brien J., Wilson I., Orton T., and Pognan 15. De Jong, D. W. and Woodlief, W. G. (1977)
F. (2000) Investigation of the Alamar Blue Fluorometric assay of tobacco leaf dehydroge-
(resazurin) fluorescent dye for the assessment nases with resazurin. Biochim. Biophys. Acta,
of mammalian cell cytotoxicity. Eur J Biochem, 484, 249–259.
267(17):5421–5426 16. Squatrito R. C., Connor J. P., and Buller R. E.
8. Perrot S., Dutertre-Catella H., Martin C., Rat (1995) Comparison of a novel redox dye cell
P., and Warnet J. M. (2003) Resazurin metabo- growth assay to the ATP bioluminescence
lism assay is a new sensitive alternative test in assay. Gynecol Oncol, 58(1):101–105
isolated pig cornea. Toxicol Sci, 72(1):122–129 17. Jonsson, K. B., Frost, A., Larsson, R.,
9. Hamid R., Rotshteyn Y., Rabadi L., Parikh Ljunghall, S., and Ljunggren, O. (1997) A
R., and Bullock P. (2004) Comparison of ala- new fluorometric assay for determination of
mar blue and MTT assays for high through- osteoblastic proliferation: effects of glucocor-
put screening. Toxicol In Vitro, 18(5): ticoids and insulin-like growth factor-I. Calcif.
703–710 Tissue Int, 60, 30–36.
