Application Note

MTT reduction - a tetrazolium-based colorimetric assay

for cell survival and proliferation
Contributed by
Joan M. Chapdelaine
Pharmakon Research International, Inc.
Waverly, PA, 18471

INTRODUCTION In 1983, a quantitative colorimetric assay for mammalian cell survival and cell prolifera-
tion was proposed by Mosmann.1 The assay is dependent on the reduction of the tetrazo-
lium salt MTT (3-(4,5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium bromide) by the
mitochondrial dehydrogenase of viable cells to form a blue formazan product. The assay
measures cell respiration and the amount of formazan produced is proportional to the
number of living cells present in culture. The assay has been shown to be a simple, rapid
alternative to counting cells by dye inclusion/exclusion, monitoring the release of 51Cr
from lysed cells, or the incorporation of [3H]-thymidine into cellular DNA. The MTT
assay has been used with a growing number of cell types including primary cultured cells
as well as established cell lines. This colorimetric microplate assay is cost effective
because of the number of tests which can be performed at one time without the problem of
radio-isotope and contaminated materials disposal.

Applications of the MTT assay

The use of the colorimetric assay for cell growth and cell survival offers major advantages
in speed, simplicity, cost, and safety over conventional cell counting assays. Figure 1
shows in a block diagram form how the MTT assay can be substituted for the thymidine
uptake assay. The MTT assay has fewer steps, uses fewer materials, and does not carry the
added burden of radioactive waste disposal.

1
The sensitivity of the MTT assay is dependent on the cell type, their average metabolic
status, and the technique selected for solubilizing the formazan crystals. For example, the
MTT assay was shown to detect as few as 200 EL4.3 cells per well and was linear to
50,000 cells per well.1 Whereas the sensitivity for T-cell blasts, which produce less forma-
zan than cell lines like EL4, was reported to be 1000 cells per well.2

Prepare tissue
culture plate with
cells and effectors

Thymidine
MTT Assay Uptake Assay
Incubate tissue
culture plate

Add MTT solution to Add H3-Thymidine to

microplate wells microplate wells

Incubate 4 hours at Incubate 4 hours at

37° C 37° C

Add formazan Harvest cells into filter

solubilizing solution paper using cell
harvester

Transfer filter paper to

scintillation vials Add
scintillation cocktail

Read tissue culture Measure radioactive

plate in MAXline incorporation with a
microplate reader scintillation counter

Process and prepare data Dispose of

summarizing the experiment radioactive
waste

Figure 1: Block diagram showing the substitution of the MTT colorimetric assay for the [3H]-thymidine uptake
assay

2
 The MTT assay, like the dye exclusion assay, must be optimized for seeding conditions
and assay duration to obtain satisfactory results. A standard curve for formazan production
as a function of the cell number and the growth rate should be determined for each cell
type. Once optimized the MTT assay has proven to be adaptable to the needs of the exper-
imental design and has been shown to be a practical method in a variety of applications:
1 Mosmann1 reported using the MTT assay to measure proliferative lymphokines, mito-
gen stimulations, and complement-mediated lysis.
2 Cell activation levels have been quantified independent of proliferation.3
3 Cell viability assays could be run more rapidly using MTT rather than counting cells
using trypan blue exclusion.4
4 This colorimetric assay had been used with anti-proliferative monoclonal antibodies,5
growth factors,2,6 and chemotherapeutic screening .7, 8
5 Tada et.al.6 showed the quantitative assay for interleukin 2 using the MTT assay to be
equivalent to the [3H]-thymidine incorporation assay. The mean of the standard errors
for the MTT assay was 3.9% as compared to 3.5% observed in the [3H]-thymidine
incorporation assay and the results from the two assays were shown to agree.

METHODS AND Table 1 is a summary of various MTT colorimetric assay methods. The MTT assay as
MODIFICATIONS developed by Mosmann1 uses the same standard 96 well flat bottom tissue culture plate
for the initial incubation of the cells and assaying for the cell number. At the end of the ini-
tial incubation, a small volume of an MTT solution was added to each well of the culture
plate. The plate was incubated to allow the formazan crystals to accumulate. The crystals
were solubilized by the addition of an acidic alcohol solution before making an optical
density measurement.
To obtain complete mixing of the acidic alcohol solution with the media, the liquid in each
wells had to be manually agitated with a pipet. The alcohol solubilized the formazan crys-
tals and the acid lowered the pH of the medium to minimize interference due to the phenol
red indicator which would interfere with the formazan reading. The yellow, acidic form of

3
the dye does not interfere. The plates were read at 570 nm with a 630 nm reference to
negate the effect of cell debris and precipitated proteins which may be produced by the
acidic alcohol addition.

T. Mosmann1 F. Denizot & H. Tada et.al.6 J M.B. Hansen, J. Carmichael, et al.7

R. Lang2 Chapdelaine et al.9
Unpublished
Sample 0.1 mL 0.1 mL 0.1 mL 0.2 mL 0.1 mL 0.2 mL
Volume
Intermediate None Remove None None None ul None
Steps medium

MTT Volume 10 µL 10 µL 20 µL 20 µL 25 µL 50 µL

Concentratio 5 mg/mL 5 mg/mL 5 mg/mL 10 mg/mL 5 mg/mL 2 mg/mL

n

MTT 4 Hours 3 Hours 4.5 Hours 4 Hours 2 Hours 4 Hours

Incubation

Adherent Nonadherent
Cells Cells

Intermediate None Centrifuge None Remove 180 None Remove all Centrifuge
Steps microplates, µL medium medium microplates,
remove remove 170
unreacted µL medium
MTT

Solubilize
Formazan

Volume 150 µL 50 µL 100 µL 180 µL 100 µL 100 µL 100 µL

Solvent Isopropanol/ Isopropanol 10% SDS in 10% SDS 20% SDS in Mineral oil DMSO
HCl 0.01 M HCl 50% DMF,
pH 4.7

Incubation 5 Minutes 1 Minute with Overnight at Overnight at Overnight at Overnight at 5 Minutes

shaking 37°C 37°C 37°C 40°C

Read 570 - 630 nm 560 - 690 nm 590 nm 570 - 650 nm 570 nm 565 nm 540 nm

Comments: •Serum •Use half-area •Use a •Higher •Improved •Improved •Less

proteins microplates2 detergent to detergent solubilization signal relative reproducible7
precipitate dissolve concentration of formazan to alcohol
with acid/ •Use serum formazan6 crystals9 solubilization7 •Signal
alcohol1,2,6 free medium •No acid to intensity
to eliminate •Good mixing precipitate •More signal •Stable color7 depends on
•Mechanical protein without added proteins relative to residual
mixing precipitation2 agitation6 acid/alcohol medium
required to •Phenol red in or acid/SDS volume7
solubilize •No phenol •Stable color6 medium is not solubilization9
formazan1,6 red2 a problem •Unstable
•Less protein since medium color7
•Plates read precipitation6 is removed
with in 1
hour1

Table 1: Summary table of MTT colorimetric assay methods.

Denizot and Lang2 found the tetrazolium dye assay as described by Mosmann to be less
sensitive than the [3H]-thymidine uptake assay for their application. They modified the
MTT method to improve sensitivity and performance. They used a microplate with a
smaller well volume, doubled the MTT concentration, and dissolved it in a serum free
media without the phenol red indicator. The unreacted MTT was removed from the centri-
fuged plate before solubilizing the formazan crystals with isopropanol. Removing the
serum proteins and the phenol red indicator reduced the background optical density and
the need for an acidic solvent, respectively. Finally, a different reference wavelength, 690
nm, was selected which increased the signal by about 25%. The authors report that these
modifications improved the sensitivity of the MTT assay 2- to 4-fold over the Mosmann
procedure.

4
 Tada et. al.6 re-examined the MTT assay in detail and modified the assay to make it possi-
ble to measure a larger number of samples at one time with less labor and with greater
accuracy. The MTT concentration was doubled and the incubation time was increased by
a half an hour. The most significant modification was in how the formazan crystals were
dissolved. In place of the acidic alcohol solution, an acidic detergent solution was substi-
tuted. This modification resolved two drawbacks with the previously described MTT
assay. First, the acidic detergent solution mixed well with the growth medium thus elimi-
nating the need to mix each well and, second, the removal of the alcohol reduced the like-
lihood of serum protein precipitation. Variations of this method have since been
developed. Chapdelaine (unpublished) removes the medium from the well of the tissue
culture plate before adding the detergent solution, thus eliminating the need to acidify the
medium. Hansen et. al.9 used a buffered detergent and added dimethyl formamide (DMF)
to improve the solubilization of the formazan crystals.
Carmichael et. al.7,8 modified the MTT assay and developed two protocols: one for adher-
ent and the other for nonadherent cell lines. For adherent cells, the media was aspirated
from the wells and mineral oil was used to dissolve the formazan crystals because it is rel-
atively nontoxic. The plates were read at 570 nm. Plates with nonadherent cells were cen-
trifuged after the incubation with the MTT. The medium was aspirated from the wells
leaving about 30 µL behind. The formazan crystals were solubilized with dimethyl sulfox-
ide (DMSO) which rapidly solubilized the formazan crystals but produced an unstable
color. The absorbance plate was read using a 540 nm filter within minutes.

CONCLUSION A problem with the MTT assay, the difficulty of solubilizing the formazan crystals with-
out precipitation of serum proteins, may be mitigated by using more effective solubilizing
agents and alternate procedures. Precipitated protein and cellular debris present in the cul-
ture plate wells are a potential source of experimental error because they interfere with the
optical readings. Dual wavelength photometry, reading the plates at a principle and a ref-
erence wavelength, is used to cancel out the effect of the debris. If the interfering material
shifts position between readings, the benefit of dual wavelength photometry is lost. The
MAXline microplate readers do not move the microplate during the read thus reducing the
chance of debris shifting. The result is better precision and an assurance that the micro-
plate reader is not a source of experimental variation.

REFERENCES 1 Mosmann, T. Rapid colorimetric assay for cellular growth and survival: Application to
proliferation and cytotoxicity assays. J. Immunol. Methods 65: 55-63 (1983).
2 Denizot, F. and Lang, R. Rapid colorimetric assay for cell growth and survival. Modi-
fication to the tetrazolium dye procedure giving improved sensitivity and reliability.
J. Immunol. Methods 89:271-277 (1986).
3 Gerlier, D. and Thomasset, T. Use of MTT colorimetric assay to measure cell activa-
tion. J. Immunol. Methods 94:57-63 (1986).
4 Penit, C. and Papiernik, M. Regulation of thymocyte proliferation and survival by
deoxynucleosides. Deoxycytidine produced by thymic accessory cells protect thy-
mocytes from deoxyguanosine toxicity and stimulates their spontaneous proliferation.
Eur. J. Immunol. 16:257-263 (1986).
5 Vaickus, L. and Levy, R. Antiproliferative monoclonal antibodies: Detection and ini-
tial characterization. J. Immunol. 135:1987-1997 (1985).
6 Tada, H., Shiho, O., Kuroshima, K., Koyama, M., and Tsukamato, K. An improved

5
 colorimetric assay for interleukin 2. J. Immunol. Methods 93:157-165 (1986).
7 Carmichael, J., DeGraff, W.G., Gazdar, A.F., Minna, J.D., and Mitchell, J. B. Evalua-
tion of a tetrazolium-based semiautomated colorimetric assay. Assessment of
chemosensitivity testing. Cancer Res. 47:936-942 (1987).
8 Carmichael, J., DeGraff, W.G., Gazdar, A.F., Minna, J.D., and Mitchell, J. B. Evalua-
tion of a tetrazolium-based semiautomated colorimetric assay. Assessment of radiosen-
sitivity. Cancer Res. 47: 943-946 (1987).
9 Hansen, M.B., Nielsen, S.E., and Berg, K. Re-examination and further development of
a precise and rapid dye method for measuring cell growth/cell kill. J. Immunol. Meth-
ods 119:203-210 (1989).

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