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Sulforhodamine B Assay and Chemosensitivity


Wieland Voigt
Summary
The sulforhodamine B (SRB) assay was developed by Skehan and colleagues to measure
drug-induced cytotoxicity and cell proliferation for large-scale drug-screening applications. Its
principle is based on the ability of the protein dye sulforhodamine B to bind electrostatically and
pH dependent on protein basic amino acid residues of trichloroacetic acidfixed cells. Under
mild acidic conditions it binds to and under mild basic conditions it can be extracted from cells
and solubilized for measurement. Results of the SRB assay were linear with cell number and cellular protein measured at cellular densities ranging from 1 to 200% of confluence. Its sensitivity is comparable with that of several fluorescence assays and superior to that of Lowry or
Bradford. The signal-to-noise ratio is favorable and the resolution is 10002000 cells/well. It
performed similarly compared to other cytotoxicity assays such as MTT or clonogenic assay.
The SRB assay possesses a colorimetric end point and is nondestructive and indefinitely stable.
These practical advances make the SRB assay an appropriate and sensitive assay to measure
drug-induced cytotoxicity even at large-scale application.

Key Words
Sulforhodamine B; trichloroacetic acid; optimal cell number; cytotoxicity.

1. Introduction
The sulforhodamine B (SRB) assay as first described by Skehan and colleagues was developed for use in the disease-orientated, large-scale anticancer
drug discovery program of the National Cancer Institute (NCI) that was
launched in 1985 (1). The SRB assay is based on the ability of the SRB dye to
bind electrostatically and pH dependent on protein basic amino acid residues.
Under mild acidic conditions, SRB binds to protein basic amino acid residues
of trichloroacetic acid (TCA)fixed cells. It can be quantitatively extracted from
cells and solubilized for optical densitiy (OD) measurement by weak bases
such as Tris base. Results of the SRB assay were linear with the number of
From: Methods in Molecular Medicine, vol. 110: Chemosensitivity: Vol. 1: In Vitro Assays
Edited by: R. D. Blumenthal Humana Press Inc., Totowa, NJ

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cells and the cellular protein measured at cellular densities in 96-well microtiter
plates ranging from 1 to 200% of confluence (1). The sensitivity of the SRB
assay is comparable with that of several fluorescence assays and is superior to
that of Lowry or Bradford. It has a signal-to-noise ratio at 5000 cells/well of
4.83 and a resolution of 10002000 cells/well (1). The end point of the SRB
assay is colorimetric, nondestructive, and indefinitely stable. The SRB assay
represents an appropriate and sensitive assay to measure drug-induced cytotoxicity and is useful to quantify clonogenicity. In addition, the SRB method
is rapid and inexpensive. In comparison with tetrazolium assays [2,3-Bis
(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) or
3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay] or
clonogenic assay, the SRB assay performed similarly when data were limited to
the inhibitory 50% concentration (IC50) (25). More important, since the endpoint measurement is not time critical, the SRB assay possesses practical advantages over the tetrazolium assays. Once automated using a microplate washer
and microplate reader, it is suitable for high-throughput screening approaches.
SRB is an anionic bright pink aminoxanthene protein dye with two sulfonic
groups. Its molecular formula and molecular weight are C27H30N2O7S2 and
558.66, respectively. The optimal wavelength for measurement of the OD of SRB
is 564 nm. Curves of OD vs dye concentration were linear up to 1.52 OD units.
When linearity range is exceeded, it is necessary to dilute an aliquot or to use
a suboptimal wavelength (490550 nm). SRB fluoresced with laser excitation
at 488 nm, and measuring SRB fluorometrically increases sensitivity about
threefold (1).
When establishing the SRB assay, Skehan et al. (1) determined the SRB
binding as a function of time, dye concentration, number of destaining washes,
and dye volume per unit area of cell culture. Based on their results, it is recommended that a 96-well culture plate be stained with 100 L of 0.4% SRB
solution per well for 30 min. The number of acetic acid washes to remove
unbound dye should be at least four (1).
Most researchers use the IC50 as a measure of in vitro cytotoxicity. This is
defined as the concentration that yields 50% less cells than the drug-free control. At the NCI, the GI50 (concentration that causes 50% growth inhibition)
was defined. The GI50 is corrected for the cell count at time zero (start of drug
exposure) and follows the following equation:
100 (T T0) / (C T0)

in which T is the OD after exposure to a certain concentration of a drug, T0 is


the OD at the start of drug exposure, and C is the OD of the control. To present
response patterns of a drug in a cell line panel such as the NCI 60 panel, a
mean graph could be constructed (6).

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The clinical relevance of in vitro cytotoxicity data is a critical issue. To estimate potential clinical activity of a drug based on in vitro data, we and others
defined the relative antitumor activity (RAA) as peak plasma concentration of
a drug/IC50 (7).
Overall, it is assumed that in vitro drug screening was approx 60% reliable
for predicting in vivo sensitivity and 90% reliable for in vivo resistance.
2. Materials
2.1. Seeding of Microtiter Plates and Drug Exposure
1. 96-Well culture microtiter plates.
2. Conventional cell culture equipment and reagents such as cell culture microscope
and cell incubator, sterile tubes (different sizes), growth medium and fetal calf
serum (according to the requirements of cultured cell lines), trypsin.
3. Cell count chamber or other cell count device such as a Coulter counter.
4. Eight-channel multipipet.
5. Sterile gauzes.
6. Sterile plastic troughs.

2.2. SRB Procedure


1.
2.
3.
4.
5.
6.
7.
8.

10% (w/v) TCA.


1% Acetic acid.
0.4% (w/v) SRB dissolved in 1% acetic acid (store at 4C protected from light).
Eight-channel multipipet.
10 mM Unbuffered Tris base (pH 10.0).
Deionized water.
Automated microplate washer.
Automated microplate reader (enzyme-linked immunosorbent assay [ELISA]
reader).

3. Methods
All cell culture work must be performed under sterile working conditions
and under standard cell culture conditions (humidified air, 5% CO2, and 37C)
or adapted to requirements of the specific cell lines, if necessary.
3.1. Seeding of Microtiter Plates for Growth Kinetics
For cytotoxicity experiments, it is critical to ensure exponential cell growth
for the entire duration of the assay. In particular, this is dependent on the
number of cells seeded per well in the microtiter plate at the initiation of the
experiment. Therefore, prior to cytotoxicity experiments, it is important to
determine the optimal cell count ensuring exponential cell growth for the entire
period of the assay and an OD at the end of the experiment in a range of
1.52.0.

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3.1.1. Day 1
1. Harvest cells by trypsinization and determine the cell count according to standard cell culture procedures (see Note 1).
2. Seed serial dilutions of cells (should cover a broad range, e.g., 10050,000 cells/
well) in microtiter plates in 100 L of growth medium per well at d 1 (keep one
eight-well row as a blank containing only growth medium, usually row 1). The
number of culture plates seeded depends on the desired length of the cytotoxicity
experiment (usually 57 d).

3.1.2. Day 2
1. Gently remove the growth medium of one culture plate (generally, we flip the
culture plate and soak up residual medium with a sterile piece of gauze while
keeping the culture plate upside down).
2. Fix cellular protein by the addition of 100 L of 10% TCA (4C) per well.
3. Add 100 L/well of growth medium to the remaining plates only at this time
point.
4. Stop another culture plate by fixating cellular protein as described in steps 1 and
2 every 24 h until all the plates are fixed. Fixed culture plates must be kept at 4C
for at least 1 h but can be stored for up to several days prior to analysis by the
SRB assay (keep in mind that storage for several days may lead to a slight
increase in background levels).

At this time point, perform the SRB assay as described next. Analyze OD
values graphically as illustrated in Fig. 1. From these growth kinetics plots,
the optimal number of cells per well can be depicted for use in further cytotoxicity experiments.
3.2. Choice of Drug Dose Range and Seeding of Culture Plates
for Cytotoxicity Assays
The design of the culture plates depends on the intended experiment. For
example, for a regular dose-response curve of a single drug, rows 1 and 12 are
kept as blank rows containing only growth medium (to determine the nonspecific background). The choice of doses to be tested and the exposure time to the
drug are the most critical points. It is conventionally agreed that a drug, when
singly administered, must be used at concentrations around the clinically
achievable peak plasma concentration in cancer patients. Dose- and timeresponse curves must also be defined for each drug in order to identify the concentration and the exposure time capable of inhibiting cell growth by 50%
(IC50). We usually do half log step dilutions, ensuring a homogeneous distribution of data points on the growth curve and coverage of a broad activity range
(see Fig. 2). This further allows extrapolation of data concerning drug dose at
high levels of activity, e.g., 90% of growth inhibition or higher.

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Fig. 1. Determination of optimal cell number per well for cytotoxicity experiments
by cell growth kinetics. A typical cell growth kinetics is illustrated. Various numbers of
cells/well were seeded in 96-well culture plates and fixed at the indicated time points.
OD was measured at 570 nm subsequent to performing the SRB assay. OD values were
evaluated graphically, and the optimal cell number for further growth experiments could
be determined from the plots. According to the two criteria, exponential cell growth for
the entire assay period and OD 1.52 at the end of the 120-h assay time, the optimal
cell number for further cytotoxicity experiments is 1600 cells/well.

3.2.1. Day 1
1. Seed in 100 L of growth medium a cell linespecific number of cells (see Subheading 3.1.) per well in 96-well culture plates. Include blanks depending on the
experimental design, and allow 24 h of cell growth.

3.2.2. Day 2

Exponentially growing cells are exposed to serial dilutions of the drug for
the desired times (we usually choose a 2- or 96-h drug exposure time).
1. Control cellular growth, distribution of cells in the well, and cellular density in
different wells using a cell culture microscope (see Note 2).
2. Add 100 L/well of drug-free growth medium to the blank and control rows (usually rows 1, 12, and 2 in our laboratory, respectively).
3. Add 100 L/well of growth medium containing different drug concentrations to
rows 311 (starting with the lowest concentration and finishing with the highest).

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4. Stop one culture plate per cell line to estimate the cell count/OD at the time of
adding the drug (zero h plate).
5. Allow the cells to grow depending on the desired drug exposure time (e.g., 96 h).
6. Remove gently the growth medium by flipping the culture plate (be cautious;
medium contains potentially toxic drugs).
7. Remove residual medium by utilizing a sterile piece of gauze while keeping the
culture plate upside down.
8. Fix the cellular protein using 100 L/well of 10% TCA (4C), and store the plates
at 4C for at least 1 h prior to analysis by the SRB assay.

In contrast to the original method as described by Skehan and colleagues, we


remove growth medium before the TCA fixation step because this significantly
reduces the signal-to-noise ratio. Cell loss is negligible and assay results are
unaffected (8).
In the case of short-term drug exposure (e.g., 2 h), follow steps 6 and 7.
Then include a washing step by adding 200 L/well of either sterile growth
medium or phosphate-buffered saline. Again perform steps 6 and 7. Subsequently, add 200 L/well of complete growth medium and allow further cell
growth for, e.g., 94 h to reach a total assay time of 96 h (see Note 3).
Depending on experience and cell line characteristics, one may risk a high
and variable cell loss during the washing procedure leading to high variations
in the experimental data. In our hands, as determined by wash kinetics, cell
loss approximates cell line dependence (1020%). However, some cell lines
might have an only weak attachment to the plastic bottom of the culture well
and, thus, might be unsuitable for this kind of experiment.
Using the washing procedure as described, one can expect a drug dilution of
about 1400 to 11000, depending on the drain of residual medium or washing
solution. Keep this in mind when using highly active drugs or cell cycle
specific drugs; the remaining substance could flaw the results. If this is a critical point in the experiment, consider including an additional washing step.
However, this may increase cell loss significantly.
3.3. SRB Procedure
The SRB assay outlined next represents the method described by Skehan
and colleagues with minor modifications. Prior to beginning the SRB assay,
cellular protein must be fixed with 10% TCA for at least 1 h at 4C.
1. Rinse TCA using an automated 96-well plate washer and add five washing cycles
using deionized water (200 L/well).
2. Sharply flick the plates over a sink and remove roughly the residual wash solution
with a piece of gauze.
3. Pipet 100 L of SRB solution into each well of the culture plate using a multichannel pipet. Allow a 30-min staining period.

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4. Remove the SRB from the culture plates with an automated plate washer by five
washing cycles using 1% acetic acid (remember to prefill the automated plate
washer with the appropriate wash solution) (see Note 4).
5. Flick the culture plates over a sink and remove the residual wash solution with a
piece of gauze.
6. Air-dry the culture plates until no further moisture is visible. (When air-dried,
both TCA-fixed and SRB-stained culture plates can be stored indefinitely. Trissolubilized SRB is also stable for extended periods of time, provided that evaporation does not occur.)
7. Solubilize the protein-bound dye with 100 L of 10 mM Tris base per well. Shake
the plates for at least 10 min on a gyratory shaker to homogenize the dye solution.
8. Measure the OD by using an automated 96-well plate reader (ELISA reader) at a
wavelength of 564 nm (see Note 5).

3.4. Data Processing and Interpretation of Results


Generally, the reproducibility of the SRB assay is high and the standard
deviation (SD) relatively low. Processing of the raw data can easily be performed by programs such as Excel or similar. It should include quality control
of the data (SD? OD in correct range? [see Note 6]), subtraction of background
staining, and subtraction of the OD at the time of adding the drug (zero h
plate). The data of each eight-well row should be averaged and the SD calculated. Finally, the percentage of growth inhibition should be calculated as the
percentage of the untreated control. We developed an Excel spreadsheet that
processes the data as outlined in one step. Subsequently, plots of the processed
data can be generated by graphic programs such as Sigma plot or similar. From
these plots, one can graphically determine growth inhibitory concentrations
such as IC50 (Fig. 2). Alternatively, mathematical curve fitting can be performed, which allows a more precise estimation of the IC50. Models used are
often based on the Hill equation. The topic of curve fitting and estimation of
drug interaction is not part of this protocols description. For extended reviews,
see refs. 9 and 10.
For clinical correlation of in vitro drug sensitivity data, we use the model of
RAA (7). Potential clinical activity can be assumed if RAA exceeds 1.
4. Notes
1. It is important to obtain and maintain a homogeneous cell suspension. Cellular
clumps/aggregates should be avoided. To disaggregate cells, it is often sufficient
to pipet up and down the cell suspension with a regular cell culture pipet. Otherwise, we sometimes use a syringe and a 26-gauge needle. If this is still insufficient, changing the trypsinization procedure might help. Depending on the cell
line used, one may have a varying fraction of dead cells. To estimate this fraction,
we usually add Trypan blue when counting the cells in a cell chamber. It is advisable to correct the cell count according to the fraction of dead cells.

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Fig. 2. Graphic determination of IC50 from dose-response plots. A typical doseresponse plot is illustrated. For graphic determination of the IC50 (drug concentration
that yields 50% less cells than the drug-free control), a bar (P) parallel to the x-axis and
intersecting the point 50% on the y-axis was constructed. In the next step, a bar was
plotted parallel to the y-axis that starts from the point of intersection of P with the
dose-response plot. The IC50 could then be directly determined at the point of intersection with the x-axis.

2. To ensure optimal assay results and a low SD, it is important to have homogeneously distributed cells in a well and an approximately similar amount of cells in
each well of each culture plate of the same cell line. Inhomogeneous cell distribution might be owing to the method of pipeting or unsuitable culture plates (the
plastic of the culture plates is a critical issue in our opinion and should be kept
constant for the entire experimental series). Sometimes it is useful to gently shake
the plates on the shelf of the incubator after seeding. If the numbers of cells vary
obviously between different wells, one should consider not continuing the experiment because of the high deviation that is to be expected. Reasons might be
an inhomogeneous cell suspension while pipeting, a malfunction of the eightchannel multipipet, a bad batch of culture plates, or an early sign of microbiological contamination.
3. Try to pipet as gently as possiblethis helps to reduce cell loss. Do not process
too many plates per time, particularly while learning the assay; one may easily run
out of time and not be able to hold the time points. Additionally, culture plates
cool down while being stored in the hood and pH of the growth medium changes

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dramatically. Sometimes, it might be beneficial to store plates on a warming


bench.
4. Carefully watch the cell monolayer at the bottom of the 96-well culture plates. If
the needles of the 96-well plate washer are poorly adjusted, they may scratch the
monolayer. This could result in partial or even complete loss of TCA-fixed cells.
Sometimes, some needles of the plate washer become clotted with debris and so
on. This may lead to inhomogeneous washing results.
5. If one does not have the appropriate filter for the microplate reader, measurements can be made at 570 nm or at a suboptimal wavelength ranging from 490 to
550 nm.
6. Usually, results of the SRB assay are highly reproducible. SD between independent experiments should not exceed 2030%. SD of an eight-well row should not
exceed 20% and is usually the highest in the range of the IC50. If the SD greatly
exceeds these values, this may be caused by additional washing steps or problems outlined in Notes 14. The OD at the end of the experiment is a critical
issue. First, measurement is not linear if OD values exceed 2. Second, if culture
wells were overgrown, this may lead to exhaustion of growth medium, partial cell
arrest, and cell death, particularly in control wells and wells with lower drug concentration. Taken together, this may lead to a significant change in IC50 (mostly an
underestimation).

References
1. Skehan, P., Storeng, R., Scudiero, D., Monks, A., McMahon, J., Vistica, D., Warren,
J. T., Bokesch, H., Kenney, S., and Boyd, M. R. (1990) New colorimetric cytotoxicity assay for anticancer-drug screening. J. Natl. Cancer Inst. 82, 11071112.
2. Rubinstein, L. V., Shoemaker, R. H., Paull, K. D., Simon, R. M., Tosini, S.,
Skehan, P., Scudiero, D. A., Monks, A., and Boyd, M. R. (1990) Comparison of in
vitro anticancer-drug-screening data generated with a tetrazolium assay versus a
protein assay against a diverse panel of human tumor cell lines. J. Natl. Cancer
Inst. 82, 11131118.
3. Perez, R. P., Godwin, A. K., Handel, L. M., and Hamilton, T. C. (1993) A comparison of clonogenic, microtetrazolium and sulforhodamine B assays for determination of cisplatin cytotoxicity in human ovarian carcinoma cell lines. Eur. J. Cancer
29A, 395399.
4. Griffon, G., Merlin, J. L., and Marchal, C. (1995) Comparison of sulforhodamine B,
tetrazolium and clonogenic assays for in vitro radiosensitivity testing in human ovarian cell lines. Anticancer Drugs 6, 115123.
5. Keepers, Y. P., Pizao, P. E., Peters, G. J., van Ark-Otte, J., Winograd, B., and
Pinedo, H. M. (1991) Comparison of the sulforhodamine B protein and tetrazolium (MTT) assays for in vitro chemosensitivity testing. Eur. J. Cancer 27,
897900.
6. Hodes, L., Paull, K., Koutsoukos, A., and Rubinstein, L. (1992) Exploratory data
analytic techniques to evaluate anticancer agents screened in a cell culture panel.
J. Biopharm. Stat. 2, 3148.

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7. Voigt, W., Bulankin, A., Muller, T., Schoeber, C., Grothey, A., Hoang-Vu, C., and
Schmoll, H. J. (2000) Schedule-dependent antagonism of gemcitabine and cisplatin
in human anaplastic thyroid cancer cell lines. Clin. Cancer Res. 6, 20872093.
8. Papazisis, K. T., Geromichalos, G. D., Dimitriadis, K. A., and Kortsaris, A. H.
(1997) Optimization of the sulforhodamine B colorimetric assay. J. Immunol.
Methods 208, 151158.
9. Berenbaum, M. C. (1989) What is synergy? Pharmacol. Rev. 41, 93141.
10. Greco, W. R., Bravo, G., and Parsons, J. C. (1995) The search for synergy: a critical review from a response surface perspective. Pharmacol. Rev. 47, 331385.

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