Limitation of the MTT and XTT assays for measuring cell viability

due to superoxide formation induced by nano-scale TiO

2
Shuguang Wang
a,
, Hongtao Yu
b
, Jeffrey K. Wickliffe
a
a
Department of Environmental Health Sciences, Tulane University, New Orleans, LA 70112, USA
b
Department of Chemistry and Biochemistry, Jackson State University, Jackson, MS 39217, USA
a r t i c l e i n f o
Article history:
Received 7 June 2011
Accepted 8 July 2011
Available online 26 July 2011
Keywords:
Tetrazolium
MTT/XTT
Superoxide
Cell viability
Nano-TiO
2
a b s t r a c t
The reduction of the tetrazolium salts, MTT and XTT, is used to estimate cell viability and proliferation.
However, superoxide can also reduce tetrazolium salts to produce the absorbant formazan end products.
Evidence indicates that nano-TiO
2
induces superoxide formation in different mammalian cells. Therefore,
studies investigating the cytological effects of nano-TiO
2
may encounter misleading results when using
MTT/XTT to measure viability or proliferation. In this study, cell viabilities of Chinese hamster ovary cells
were assayed using MTT, XTT and the trypan blue exclusion assay following exposure to nano-TiO
2
. In
comparison to the trypan blue exclusion assay, the MTT and XTT assays inaccurately predicted cell tox-
icity or overestimated cell viability respectively. XTT, in particular, appears more sensitive to superoxide
than MTT. The reduction rate of XTT is 1.5 times that of MTT and SOD inhibition of XTT is less effective
than that of MTT, indicating that XTT is more reactive with O

2
than MTT. Therefore, using XTT or MTT for
measuring cell viability or proliferation may yield inaccurate results when conditions in cultured cell
increase superoxide formation.
2011 Elsevier Ltd. All rights reserved.
1. Introduction
The tetrazolium salts, MTT, 3-(4,5-dimethyl-2-thiazol)-2,5-di-
phenyl-2H-tetrazolium bromide, and XTT, 2,3-bis[2-methoxy-
4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide, are wide
ly used test methods to measure cell viability and proliferation
(Berridge et al., 2005; Hasen and Bross, 2010; Tsukatani et al.,
2009; Wang et al., 2010). Viability and proliferation are based on
the capacity of mitochondrial dehydrogenase enzymes present in
living cells to reduce MTT to an insoluble, purple formazan product
(Abe and Matsuki, 2000), or XTT into a water soluble, orange for-
mazan product (Roehm et al., 1991). The amount of the formazan
product is measured using a spectrophotometer to estimate rela-
tive cell viability. It is widely held that this conversion occurs only
in viable cells (Zhu et al., 2004).
However, this method can be inuenced by various conditions
including acidic pH (Johno et al., 2010), polyphenols (Han et al.,
2010), pyruvate analog (Ganapathy-Kanniappan et al., 2010), and
nanomaterials (Gormley and Ghandehari, 2009; Park et al., 2009).
MTT (Madesh and Balasubramanian, 1997, 1998) and XTT (Shi-
mamura et al., 2000; Sutherland and Learmonth, 1997) can also
be reduced by O

2
. Toxicity testing of engineered nanomaterials
including nano-TiO
2
has generated a tremendous number of publi-
cations in the last several years. Conicting cell viability and prolif-
eration results have been reported for these studies using the MTT
assay (Belyanskaya et al., 2007; Kroll et al., 2009; Plumb, 1999; Qu
and Zhang, 2010). Since nano-TiO
2
increases the formation of O

2
in
different mammalian cells (Gurr et al., 2005; Long et al., 2006,
2007), we hypothesize that MTT and XTT assays underestimate
cytotoxicity by overestimating cell viability.
Therefore, in this report, MTT and XTT assays are used to eval-
uate viabilities of Chinese hamster ovary cells (CHO-K1) after they
are exposed to nano-TiO
2
(anatase form). CHO cells have recently
been used to study gene expression, toxicity screening, cell biology,
and virology (Kang et al., 2007; Stewart and Harris, 2003) The MTT
and XTT assay results are compared to trypan blue exclusion assay,
a direct test for cell viability (Altman et al., 1993). To compare the
effect of O

2
on MTT and XTT, the reduction of MTT and XTT by O

2
generated through xanthinexanthine oxidase (XXO) will also be
examined.
0887-2333/$ - see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tiv.2011.07.007
Abbreviations: MTT, 3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2H-tetrazolium
bromide; XTT, 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-car-
boxanilide; CHO-K1, Chinese hamster ovary cells; Nano-TiO
2
, nano titanium
dioxide; SOD, superoxide dismutase; O

2
, superoxide; (XXO), xanthinexanthine
oxidase; WST, water soluble tetrazolium; NBT, nitroblue tetrazol.

Corresponding author. Tulane University School of Public Health and Tropical

Medicine, Department of Environmental Health Sciences, 1440 Canal Street, Suite
2100, New Orleans, LA 70112, USA. Tel.: +1 504 988 9046.
E-mail address: swang4@tulane.edu (S. Wang).
Toxicology in Vitro 25 (2011) 21472151
Contents lists available at ScienceDirect
Toxicology in Vitro
j our nal homepage: www. el sevi er . com/ l ocat e/ t oxi nvi t
2. Materials and methods
2.1. Materials
CHO-K1 cells were obtained from the ATCC (Manassas, Vir-
ginia). Fetal bovine serum (FBS), Hams F12, L-glutamine, penicil-
lin/streptomycin, trypan blue and 0.05% trypsin-EDTA solution
were from Invitrogen (Frederick, MD). Titaniumdioxide (100% ana-
tase, <25 nm), XTT, MTT, xanthine and xanthine oxidase were from
SigmaAldrich (St. Louis, MO). Dimethyl sulfoxide (DMSO) and
phosphate buffered saline (PBS) were from Fisher Scientic (Hous-
ton, TX).
2.2. Cell culture and treatment
CHO-K1 cells were cultured in Hams F12 medium supple-
mented with 10% FBS, 1% L-glutamine, and 1% penicillin/strepto-
mycin. Cells were cultured in 25 cm
2
culture asks in a
humidied incubator at 37 C with 5% CO
2
. After the CHO-K1 cells
grew to the expected concentration, they were harvested by tryps-
inizing the cell with 0.05% trypsin/EDTA and incubated at 37 C for
5 min to obtain the complete cell detachment. The cell suspension
was centrifuged at 200 rpm for 5 min, cell pellets were resus-
pended with complete medium. The cell suspension was counted
and adjusted to 2.5 10
5
cells/ml. The cell suspension with differ-
ent concentration of nano-TiO
2
(0, 25, 50, 100 and 200 lg/ml) were
added into the wells of 96-well plate for XXT and MTT assay, or
60 mm dishs for trypan blue exclusion assay. The treated cells
were incubated for 24 h and 48 h with 5% CO
2
at 37 C before
assays.
2.3. XTT and MTT assays
After 24 h and 48 h nano-TiO
2
treatment, cell viabilities were
assessed using MTT and XTT according to the manufacturers pro-
tocol (Sigma). Six replicates for each exposure concentration were
examined. To eliminate background absorption, the plates were
centrifuged at 200 g for 5 min and the supernatant of each well
was transferred to a new plate. Absorbance values at 570 nm for
MTT and 450 nm for XTT (corrected at 690 nm) were measured
using a microplate spectrophotometer (Bio-RAD Benchmark Plus).
2.4. Trypan blue exclusion assay
After 24 h and 48 h nano-TiO
2
treatment, cells were washed
with PBS buffer twice and trypsinized. The cell suspension was
centrifuged at 200 g for 5 min and the supernatant of each dish
was removed. The cell pellet was resuspended in 1 ml PBS buffer.
The cell suspension (100 ll) was stained with 50 ll of trypan blue
(0.4%). Cell counts and viabilities were determined visually using a
hemocytometer and light microscope.
2.5. MTT and XTT reduction by XXO and inhibition by SOD
A solution containing 50 ll of 3 mM xanthine, 50 ll of 3 mM
EDTA, and 50 ll of 1 mg/ml XTT or MTT was added to 800 ll of
PBS buffer (pH 7.4). SOD was added to control samples at a concen-
tration of 10 lg/ml. The reaction was triggered by the addition of
50 ll of 1 mg/ml XO solution. Absorbance during the rst 10 min
at 450 nm for XTT assay and 570 nm for XTT was recorded by
NANODROP 2000c (Thermo Scientic) at 25 C. To measure SOD
inhibition, SOD was added in separate experiments at a nal con-
centration of 0, 0.04, 0.2, 1, and 5 lg/ml. The inhibition reaction
was initiated by the addition of 50 ll of 1 mg/ml XO solution.
Absorbance was measured as previously described.
2.6. Statistics
Indicator absorbance values at 24 or 48 h time points for both
MTT and XTT were analyzed using a general linear model and post
hoc arithmetic mean comparisons that were Bonferroni-corrected.
Cell counts and viabilities using the trypan blue exclusion assay
were analyzed in the same manner. An alpha level of p < 0.05
was used as the criterion for statistical signicance. The SPSS ver.
16.0 for Windows program was used for statistical testing (IBM
Corporation; Somers, NY).
3. Results
CHO-K1 cells were treated with 0, 25, 50, 100 and 200 lg/ml of
nano-TiO
2
for 24 h or 48 h. The MTT, XTT and trypan blue exclusion
assays were used to determine cell viability as shown in Fig. 1.
Based on the trypan blue assay, no decrease in cell viability was
noted up to 50 lg/ml of nano-TiO
2
, but viability decreased at the
two higher nano-TiO
2
concentrations of 100 and 200 lg/ml. The
decrease in cell viability using the MTT assay was only observed
at the highest nano-TiO
2
concentration of 200 lg/ml at 48 h incu-
bation. The ratios of cell viability for MTT/trypan blue assays are
1.09 and 1.33 for 24 h incubation and 1.15 and 1.85 for 48 h incu-
bation in the 100 and 200 lg/ml of nano-TiO
2
groups, respectively.
In comparison to the trypan blue exclusion assay, the MTT assay
did not detect a decrease in cell viability at concentrations at and
above 100 lg/ml of nano-TiO
2
. The XTT assay appeared to overes-
timate cell viability at all tested concentrations. The ratios of cell
viability for the XTT/trypan blue assays are 1.53, 1.82, 2.38 and
2.46 for 24 h incubation and 1.48, 1.56, 2.86 and 2.92 for 48 h incu-
bation in the presence of 25, 50, 100 and 200 lg/ml of nano-TiO
2
,
respectively. This suggests that the MTT and XTT assays failed to
Fig. 1. Dose-dependent CHO cell viability after exposure to nano-TiO
2
for 24 h and 48 h determined by trypan blue exclusion (A), MTT(B), or XTT(C) assays. The results were
expressed as percent of the reading in control wells. The mean S.D are two independent experiments. Asterisks indicate signicant difference (GLM-ANOVA, Bonferroni-
corrected) from respective time point control (p < 0.05).
2148 S. Wang et al. / Toxicology in Vitro 25 (2011) 21472151
detect a decrease in cell viability in relative comparison to the try-
pan blue exclusion test in the presence of nano-TiO
2
, and XTT is
more sensitive to superoxide than MTT.
To test this in the absence of cells and independent of nano-TiO
2
,
XXO were used to generate O

2
. SOD was used to quench acellular
production of O

2
. The reaction rate of MTT and XTT with O

2
gener-
ated by XXO was determined (Fig. 2A). A zero order reaction was
observed with rate constants of 0.0817 for XTT (R
2
= 0.995) and
0.055 for MTT (R
2
= 0.999). The ratio of the rate constants for XTT/
MTT is 1.5, indicating that XTT is more reactive with O

2
than MTT.
This result is conrmedin Fig. 1 where the XTT assay indicatedhigh-
er, nano-TiO
2
-dependent cell viabilities than the MTT assay. SOD
inhibitionof formazanproductionfromXTTis less efcient thanthat
fromMTT. SODadded to the XTT/XXOor MTT/XXOsysteminhib-
ited formazan formation in a concentration-dependent manner
(Fig. 2B). The reaction of O

2
with SOD is rst order with respect to
O

2
concentration (Heinrich et al., 2006). Thus, SOD competes with
XTT or MTT for O

2
. At lower SOD concentrations of 00.2 lg/ml,
SOD competes more favorably with the slower reacting MTT, thus
increasing inhibition. At higher SOD concentrations of 1 and 5 lg/
ml, the SOD reaction with O

2
dominates, inhibiting nearly 95% of
the reaction of O

2
with both MTT and XTT.
4. Discussion
Toxicity testing of engineered nanomaterials (ENMs) including
nano-TiO
2
has generated a tremendous number of publications in
the last several years. Conicting cell viability and proliferation re-
sults have been reported in ENM studies using the MTT assay (Kroll
and Pillukat, 2009; Plumb, 1999), especially on carbon nanomate-
rials (Belyanskaya and Manser, 2007; Qu and Zhang, 2010). Due to
the importance these tetrazolium salts play in biological testing,
and the importance of nano-TiO
2
, it is worth examining factors
inuencing the results of these viability assays to avoid future
controversies.
Fig. 2. A: Time dependent XTT and MTT reduction by superoxide generated from xanthine and xanthine oxidase reaction, 10 lg/ml of SOD in the controls. B: Concentration of
SOD-dependent inhibition of the reaction of XTT and MTT with superoxide generated by xanthine/xanthine oxidase. Absorbance was recorded at 570 nm for MTT and 450 nm
for XTT.
MTT
MTT formazan
N
N
N
N O
HN
H
3
CO
NO
2
SO
3
-
H
3
CO
SO
3
-
NO
2
N
N
N
N O
HN
H
3
CO
NO
2
SO
3
-
H
3
CO
SO
3
-
NO
2
H
3
CO
NO
2
SO
3
-
H
3
CO
SO
3
-
NO
2
N
N
NH
O
HN
N
N
S
CH
3
CH
3
N N
N N
N
S
CH
3
CH
3
N N
N N
N
S
C
CH3
CH3
N
N N
NH
+ e
-
+ e
-
O
2
-.
- e
-
-e
-
XTT
Intermediate radical
Intermediate radical
XTT formazan
+ e
-
+ e
-
O
2
-.
+ H
+
+ H
+
Fig. 3. Schematics of superoxide induced XTT/MTT reduction.
S. Wang et al. / Toxicology in Vitro 25 (2011) 21472151 2149
The percent CHO-K1 cell viability after exposure to nano-TiO
2
for 24 h and 48 h determined are assay dependent, with a trend
of XTT > MTT > trypan blue exclusion, especially for the XTT assay
at higher TiO
2
concentrations and longer exposure times. Some
of the XTT assay results are 23 times higher than trypan blue as-
say. This indicates that the XTT and MTT assays can overestimate
cell viability or fail to detect a decrease in cell numbers and viabil-
ity. This phenomenon is likely caused by O

2
induced by nano-TiO
2
(Gurr and Wang, 2005; Singh et al., 2007). The O

2
induced by nano-
TiO
2
results in the conversion of XTT and MTT to their correspond-
ing formazan salts to produce misleading viability results. The
reduction rate of XTT by O

2
is 1.5 times that of MTT, while SOD
inhibition of XTT is less effective than that of MTT. We hypothesize
that the mechanism of XTT or MTT reduction by O

2
is similar to
that previously proposed for the WST and NBT assays (Bielski
et al., 1980; Oritani et al., 2004). This reduction is believed to fol-
low a two-step reaction. First, an intermediate radical is formed
via one-electron transfer from O

2
. This tetrazolinyl radical will
then disproportionate to the corresponding tetrazolium salt and
formazan. The second reaction may also consist of a one electron
reduction to the formazan product. Therefore, we propose that
MTT and XTT follow the reaction as shown in Fig. 3 below.
It is known that the redox potential of tetrazolium salts is highly
dependent on their structure and substituents (Kato et al., 1995;
Marques et al., 1995). Thus, it is safe to assume that MTT, XTT,
WST, and other tetrazolium dyes used currently for toxicity testing
may have different reaction rates with superoxide. Depending on
the concentration of SOD or other molecules that also react with
superoxide in cellular, these competitive reactions may well inu-
ence estimates and interpretations of viability and proliferation.
Therefore, tetrazolium dye assays for cell viability and cell prolifer-
ation may not be as accurate and reliable if experimental conditions
inuence levels of O

2
in biological systems. Many recent reports
suggest that many nanomaterials, especially nanometal oxides,
generate ROS including O

2
in biological systems (Jin et al., 2010;
Lipovsky et al., 2011; Shukla et al., 2011; Voinov et al., 2011; Xue
et al., 2010). Therefore, caution should be exercised when using
the MTT or XTT assays to estimate viability and infer cytotoxic ef-
fects. We suggest that researchers using these assays under such
conditions independently verify viability and proliferation esti-
mates based on different endpoints (e.g. cell membrane damage
assays).
Conict of interest
None declared.
Acknowledgment
This work was supported by the Tulane University School of
Public Health and Tropical Medicine and the Tulane Cancer Center.
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