3rd International Conferences and Workshops on Basic and Applied Sciences 2010

ISBN: 978-979-19096-1-7

Evaluation of Hyperthermia Effect on Cell Viability by a

Simple Crystal Violet Method of MDA-MB 231 Cell Line
E.Asita and H.Salehhuddin
Department of Biological Science,
Faculty of Bioscience and Bioengineering,
University of Technology Malaysia, Johor, Malaysia
e-mail: asitaelengoe@yahoo.com
email: saleh@fbb.utm.my

triphenylmethane dye which taken up by cells

(Jorge et al., 2007). This technique has been used
with modifications for a wide number of
applications, most of them to determine effect of
chemicals, drugs, or toxins from pathogens on
cytotoxicity or cell death (Harhaji et al., 2004;
Shaik et al., 2004; Rothman, 1986 (tdk ad dlm
dapus)), and to evaluate cell viability (Thomas et
al., 2004) or proliferation of cell (Zivadinovc et al.,
2005) under different conditions.

Abstract
Viability of cultured mammalian cells is evaluated
by a variety of techniques. In this study, a simple
crystal violet staining method was used to
determine hyperthermia effect on viability of
human breast cancer MDA-MB 231 cell. The cells
were exposed to heat at 42C for 2 hours to estimate
the percentage of cell viability using crystal violet
procedure (4% paraformaldehyde fixation). The
value assay was measured continuously from day 1
to day 7. The percentage of viability was
significantly decreased after 7 days of heat
exposure at 42C (74.111.11%) compared to
control (100% viability) (p< 0.05). More cells were
killed at high temperature (42C) and longer
duration of heat. However, temperatures above
42C and longer exposure of heat (> 2 hours) can
cause damages in healthy cells. This study revealed
that crystal violet was a sensitive assay for adherent
cell. It is also very effective; simple; and rapid
quantitative assay to measure the cell viability of
MDA-MB 231 cell.

The most common method used with adherent cells

has been the crystal violet assay when compared to
other procedure such as trypan blue method which
the cells have to be physically or enzymatically
removed from the growth surface before measure
the cell viability (Sanford et al., 1951). Therefore,
crystal violet staining technique is very effective
because it is simple; reproducible assay of
cytotoxicity; and a number of materials can be
evaluated simultaneously (Itagaki et al., 1991;
Saotome et al., 1989). Hyperthermia means that
body tissue is exposed to high temperatures (up to
45C). It is a type of cancer treatment (Samia et al.,
2007). Research has shown that high temperatures
can damage or kill cancer cells with usually
minimal injury to normal tissue (Van der zee,
2002). In the present study, it was aimed to
determine the effect of hyperthermia on viability in
human breast cancer MDA-MB 231 cell.

Keywords: Cell viability; Crystal violet assay;

Hyperthermia; Sensitive assay.
1

Introduction

A variety of methods have been used for

evaluating the cytotoxic effects of cultured cells,
depending on the cell or tissue type. The assays
widely used in mammalian cell are based either on
colorimetric method or microscopic analysis (Juan
et al., 2003). One of the techniques, crystal violet
(CV) staining assay is a well known quantitative
colorimetric
procedure.
The
colorimetric
determination of the stained cells is based on loss of
ability to maintain and provide energy for
metabolic growth and function (Chiba et al., 1997).
Crystal violet or gentian violet is a

2 Methodology
2.1
Cells
The human breast cancer cell line MDA-MB 231
was obtained from American Type Culture
Collection (ATCC) and cultured in Dulbecco's
modified Eagle's medium supplemented with 4.5
g/L glucose, 1.5 g/L sodium bicarbonate, 100 U/mL
penicillin, 100 mg/mL streptomycin, and 10% fetal

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Asita, Evaluation of Hyperthermia Effect on Cell Viability by a Simple Crystal Violet Method of MDA-MB 231
Cell Line

assay was measured continuously from day 1 to day

7. The percentage of viability was significantly
decreased after 7 days of heat exposure at 42C
(74.111.11%) compared to control (100%
viability) (p< 0.05). More cancer cells were killed
at high temperature (42C) and longer duration of
heat with minimal damage to normal cells. This
shows that hyperthermia had a significant effect on
cell viability of MDA-MB-231. However,
temperatures above 42C and longer exposure of
heat (> 2 hours) can cause damages in healthy cells.

calf serum (FCS). Cells are maintained in a 5% CO2

humidified incubator at 37C.
2.2
Hyperthermic exposure
MDA-MB 231 cells, 1104 cells/well in 200l
culture medium, were seeded in each well of 96well plates and were pre-cultured overnight
incubation at 37C.Then, hyperthermic exposure
was performed by placing culture plates in an
incubator maintained at 42C. Well temperature
was monitored and maintained within 0.1C during
the treatment period. Cultured cells were
maintained at 37C served as controls for all
experiments.

Table 1: Viability of MDA-MB 231 cell line

exposed to heat shock treatment at 42C for 2 hours
as measured with crystal violet staining procedure
(4% paraformaldehyde fixation). The data are
presented as the mean SD (SD was within 5% of
the mean) of triplicate observations from three
independent experiments.

2.3
Crystal Violet Assay
Cell viability was measured by the crystal violet
staining method. After hyperthermic exposure, the
cultivation medium was removed gently from the
wells and cells were washed with PBS. Nonadherent cells were washed off and remaining cells
were fixed with 200l of 4% paraformaldehyde for
30 min. After washing, 100 l of 0.05% crystal
violet (CV) solution in 20% ethanol was added and
cells were allowed to stain for 30 min. Following
three washes with distilled water, the plates were
aspirated and allowed to air-dry at room
temperature. To each well, 200l of 10% acetic
acid was added and incubated for 20 min with
shaking. 100 l of the dissolved dye solution was
taken out and diluted in (1:4) distilled water. The
optical density at 570nm at each well was measured
on a microplate reader. 10% acetic acid was used as
blank. The average absorbance of the control cells
exposed to free culture medium was set to represent
100% of viability and the results were expressed as
percentage of these controls (Ito, 1984).

Day
1
2
3
4
5
6
7

2.4
Statistical analysis
Results were expressed as a mean standard error
of the mean (SEM). The mean values were
calculated from data taken from three different
experiments performed in triplicates on separate
days using freshly prepared reagents for all cases.
Significance testing was performed where indicated
using an independent t-test (Students; 2
populations) and an analysis of variance (ANOVA).
The differences were evaluated significant at p <
0.05(5%).
3

Percentage of cell viability (%)

91.004.82
87.283.59
85.804.36
83.694.96
82.032.82
76.550.60
74.111.11

Figure 1: After 2 hours of hyperthermic exposure,

percent cell viability of MDA-MB 231 was
determined by crystal violet staining technique with
4% paraformaldehyde fixation. The values assay
was measured from day 1 to day 7. The data are
presented as mean SD (SD was within 5% of the
mean) from three independent experiments in
triplicate.

Result

Effect of hyperthermia on cell viability of MDAMB 231 was evaluated using crystal violet staining
with fixation of 4% paraformaldehyde. Cells were
treated at heat 42C for 2 hours to determine the
percentage of cell viability. The results were
summarized in Table 1 and Figure 1. The value

The crystal violet assay involves simultaneous cell

lysis and the released nuclei to stain purple in a
hypertonic solution (citric acid) (Berry et al., 1996).
The stained nuclei are measured by a microplate
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3rd International Conferences and Workshops on Basic and Applied Sciences 2010

tuberculosis strains. Journal of Medical

Microbiology, 56: 733-737, 2007.

reader at absorbance 595nm. This method has been

widely used for monitoring mammalian cell
cultures because of the fast and quantitative release
of stained nuclei. Percentage viability of MDA-MB
231 cell line determined by the crystal violet
method; was significantly and consistently high (p<
0.05). Therefore, it proved that crystal violet was a
sensitive assay for adherent cell. It is also very
effective; simple; and permits many samples to be
analyzed rapidly and simultaneously (Itagaki et al.,
1991; Saotome et al., 1989).
4

[7]

Juan, M. Capasso, A, Belen, R. Cosso,

B, Tomas Berl, A, Christopher, J. Rivard,
A, Carlos, Jimenez, B., A colorimetric
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[8]

Samia, I., Mostafa, Abd, E., Hady, A.,

Abd, E.W., Gamal, M., Abu, Sinna., and
Amal, I.H., The relationship between
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carcinoma cell lines. J.Med.Lab.Sci, 16(1):
27-42, 2007.

[9]

Sanford, K.K., Earle, W.R., Evans, V. J.,

Waltz, H.K. and Shannon J.E., The
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[10]

Saotome, K., Morita, H. and Umeda, M.,

Cytotoxicity test with simplified crystal
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[11]

Shaik, M. S., Chatterjee, A. and Singh, M.,

Effects of monensin liposomes on the
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Thomas, M., Finnegan, C. E., Rogers, K.

M., Purcell, J. W., Trimble, A., Johnston,
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Conclusion

In this study, it revealed that crystal violet was a

sensitive and useful assay for adherent cell. Besides
that, hyperthermia enhanced reduction in viability
of MDA-MB-231 cell.
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ISBN: 978-979-19096-1-7

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