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CHAPTER16MTT AssaysRosa Supino1. Introduction

The MTT calorimetric assay determines the ability of viable cells toconvert a soluble tetrazolium salt [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) into an insoluble formazan precipi-tate. Tetrazolium salts accept electrons from oxidized substrates orappropriate enzymes, such as NADH and NADPH. In particular, MTT isreduced at the ubiquinone and cytochrome b and c sites of the mitochon-drial electron transport system and is the result of succinate dehydroge-nase activity. This reaction converts the yellow salts to blue-coloredformazan crystals that can be dissolved in an organic solvent whose con-centration can be spectrophotometrically determined (Fig. 1). Owing tothe many advantagesof the assay, t is today considereda significant advanceover traditional techniques. In fact, it is rapid, versatile, quantitative, andhighly reproducible with a low intratest variation between datapoints(+15% SD); it is useful in a large-scale, antitumor drug-screening pro-gram (1-3). Moreover, the test can also be used for floating cells, such asleukemias and small cell lung carcinoma, and always allows sufficienttime for cell replication, drug-induced cell death, and loss of enzymaticactivity, which generates he formazan product from the MIT substrate 4).The MTT assay has to be set up for each cell line. The optimal cellnumber seeding (since cells during the assay have to be actively metabo-lizing), the duration of the experiment, and the time of MTT incubationnecessary for an evaluable final optical density need to be selected inpreliminary experiments.

From: Methods m Molecular Biology, Vo l 43: In V/fro ToxrCrty Tes ting P rotocolsEdlted by: S. OHare and C K At&tw ill Copynght Humana Press Inc , Totowa, NJ137


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138 Supino160,

1.20.x?:. ! !

za.s 060.0

0.40 *

ooo- , I I200 300 400 500 600 700

Wavelength (nm)

Fig. 1. Absorption spectraof MTT formazan reagent 25 ug/mL) in DMSO(A) and 0.04N HCVisopropanol (B).A number of key points are relevant for the choice of the MTT assay.It is valid for a number of cell lines derived from a broad spectrum ofsolid tumors by a variety of isolation techniques. Most cell lines tested(106/l 11) exhibited acceptable calorimetric profiles (control growthabsorbance > 0.500 U). Measurements of cell growth by MTT reduc-tion correlated well with indices of cellular protein and viable cell number.At specific culture conditions and appropriate assay parameters, itprovides reproducible indices of drug sensitivity. It is very sensitive since32 human cells/well give an optical density of 0.05 U (optical densitydeveloped by murine cells is lower than that of human cells) (Fig. 2).It is comparable with other in vitro methods of drug cytotoxicityevaluation (Fig. 3).

2. Materials1. Cells: exponentially growing cell line with recognizable evels of mito-

chondrial activity.2. 96-well tissue culture plates,U-bottomed or flat bottomed.3. Scanning multiwell spectrophotometer.4. Automatic plate shaker.5. Incubater, 37OC,humidified, 5% CO2atmosphere.


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MTT Assays 139

cel l number (~10~)

Fig. 2. Relationship between cell number and optical density in B16V mela-noma cells (0) and N592 small cell lung carcinoma (A).6. Centrifuge.7. 50 mL tubes.Make up the following:

8.9. Drugs made up to solutions 10x more concentrated than required.Hanks salt solution, composition (in g/L): KC1 (0.4), KH2P04 (O-06), NaCl(8), NaHCO, (0.35), Na2HP04. 7H,O (0.09).MTI solution: 5 mg/mL in Hanks salt solution. Note: MIT solution maybe stored at 4C for about 1 wk.Culture medium, dependent on the cell line.Dimethylsulfoxide (DMSO).RPMI-1640.Fetal calf serum.0.45 p filters.Agarose.HCl.Isopropanol.Collagenase.Hyaluronidase.Ficoll-Hypaque.

10.11.12.13.14.15.16.17.18.19.20.21.22. Percoll.


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0 1 2 3 4 3 " 20 40 60 60 100 120Adrmm ycin (IN) ctsplatln (UM)

Supino-0 \*

\ sPo*+ 0A

I : : : : : 10 5 20 25 30Fig. 3. Clonogenic assay and MTT assay curves: effect of adriamycin,cisplatin, and vinblastine on V79 cells. 0 MTT assay;A Clonogenic assay.

3. Methods3.1. MTT Method

For the chemosensitivity test, exponentially growing cells were har-vested, counted, and inoculated (at the appropriate concentrations in avol of 100 pL) into 96-well microtiter plates; 8 replicates were preparedfor each dose. U-bottom microplates were used for suspension-growingcells, whereas flat-bottom microplates were used for plastic-adherent cellcultures. Immediately or 24 h after cell seeding, 10 PL of different dilu-tions of drugs, prepared 10x more concentrated than requested, wasadded to each well. After different incubation times at 37C in a humidi-fied 5% CO, atmosphere, the MTT assay was performed. MTT (Sigma,


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MTT AssaysSt. Louis, MO) was dissolved at a concentration of 5 mg/mL inHanks salt solution and filtered with a 0.45 l.~ ilter (in order to avoidMTT aggregates). Ten microliters of MTT solution was added to eachwell and also to the control wells without cells. In fact, additional con-trols consisted of media alone with no cells, with or without the variousdrugs. After 4-6 h of incubation, microtiter plates were centrifuged at2000 rpm for 10 min; medium was then removed, and 100 PL of DMSOwas added to each well. After thorough mixing with a mechanical platemixer, absorbance of the wells was read in a scanning well microcultureplate reader at test and reference wavelengths of 550 and 620 nm, respec-tively, that are approximately the peak and the lowest MTT wavelengthsof absorption required to avoid quenching from growth medium, in parti-cular phenol red. Absorbance values from all wells were corrected againstthese control absorbance levels, and the IDS0 was defined as the concen-tration of drug that produced 50% reduction of absorbance comparedwith untreated control cells.

1. Harvest, count, and noculate, n 100 uL of complete medium, the appro-priate number of cells.2. Add 10 pL of drug solution 10x more concentratedhan requested.3. Incubate or different times in a humidified atmospheren 5% CO2at 37C.4. Add 10 FL of MTT solution (5 mg/mL).5. Incubate 4-6 h in 5% CO2 at 37C.6. Centrifuge the plates at 2000 rpm for 10 min.7. Remove the medium.8. Add 100 pL of DMSO.

9. Mechanically mix the plate until formazan crystals are dissolved.10. Read in a microculture plate reader at test and reference wavelengths of550 and 660 nm, respectively.11. Calculate the mean of the optical density of different replicates of the samesample.12. Evaluate the percentage of each value vs the control.13. Plot in a semilog chart the percentage of optical density against drug con-centration.14. Determine the ID5e from the dose-response curve.3.2. HTCA-MTT Method

Different authors have reported that the assay yields results very simi-lar to the clonogenic tumor cell assay in Chinese Hamster Ovary, lung


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142 Supinocancer cells, and mouse melanoma cell lines and their derived pleiotropicdrug-resistant counterparts (5). However, data obtained with one tech-nique should be compared only with data obtained with the same tech-nique and the same treatment schedule.The MTT assay is successfully used, since other valid tests such asHTCA (human tumor colony assay) often have several technical prob-lems, such as low plating efficiency and longer assay time. However, theuse of the MTT assay for drug sensitivity testing of tumor samples mayresult in a higher background, because contamination by normal cellsmay reduce tetrazolium dye as well. To reduce such contamination, anHTCA-MTT mixed test was thus set up.After treatment for 1 h with the drugs, samples were seeded (in 96-well microtiter plates prefilled with a 50 PL underlayer of RPMI-1640and 15% fetal calf serum [FBS] in 0.4% agarose) in 50 PL of RPMI-1640 and 15% FBS in 0.25% agarose.Followmg incubation for l-7 d at37C in 5% COZ, 10 ltL of MTT (5 mg/mL) was added to each well, andthe plates were incubated at 37C for 4 h. Then, 100 ~JL of 0.04N HCI inisopropanol was added to each well, and the solution was mixed vigor-ously to solubilize the formazan product. Air bubbles were sucked out,and after 1 h at room temperature, the absorbance of the wells was mea-sured in a 2-wavelength microplate photometer at test and referencewavelengths of 550 and 660 nm, respectively.

1. Fill 96-well microplates with 50 l&L of complete medium in 0.4% agarose.2. Allow agarose to solidify.3. Add 50 ~.LL f complete medium contammg the appropriate number ofdrug-treated cells in 0.25% agarose.4. Incubate for l-7 d in a humidified atmosphere of 5% CO2 at 37C.5. Add 10 FL of MIT (5 mg/mL).6. Incubate for 4-6 h at 37C in 5% CO*.7. Add 100 l.tL of 0.04/V HCI m isopropanol.8. Mix vigorously.9. Leave for 1 h at room temperature.10. Read m a microplate photometer at test and reference wavelengths 550and 660 nm, respectively.11. Calculate the mean of the optical density of different replicates of the samesample.12. Evaluate the percentage of each value vs the control.


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MTT Assays 14313. Plot in a semilog chart the percentage f optical density againstdrug con-centration.14. Determine the IDS0 rom the dose-responseurve.The HTCA-MTT hybrid assay is successful, since the HTCA assay istime-consuming, complicated, costly, and applicable for a limited num-ber of human solid tumors. However, the MTT assay results in a highbackground owing to contamination by normal cells (especially in solidtumors), which may reduce tetrazolium dye as well. Moreover, it hasbeen reported that tumor tissues are more sensitive to various antitumordrugs than are adjacent normal mucosal tissues. In contrast, the HTCA-MTT hybrid assay is much shorter than the HTCA assay (4 d vs 2-3 wk)and is also effective to measure cells with low colony-forming efficiency.Furthermore, normal cells (fibroblasts, lymphocytes, and endothelialcells) do not grow in the double layer of agarose.

3.3. Purification of Tumor CellsOther authors have reported a technique for purification from normalcells of tumor cells using discontinuous Ficoll-Hypaque and Percoll gra-dients. Using this method they obtained a higher correlation between invitro results and clinical response (6) (Fig. 4).Tumor cells were dispersed in complete medium containing collage-nase (2 mg/mL, type V-S; Sigma, hyaluronidase (10 U/r& type IV-S;Sigma), and DNase-1 (0.4 mg/rnL; Sigma). After a 40-min incubation at37OC, the cells were harvested, washed, and suspended in completemedium. In the case of ascites, cells after centrifugation at 400g for 5

min were suspended in complete medium. Cells were then centrifugedon Ficoll-Hypaque (specific gravity 1.077, Pharmacia, Uppsala, Sweden)gradients at 400g for 30 min in 50-mL tubes (400g at the bottom of tubes).Mononuclear and tumor cells at the interface were collected, washed,and suspended at 106/mL in complete medium. The cells were then lay-ered on discontinuous gradients consisting of 10 mL of 100% and 15 mLof 75% Ficoll-Hypaque in 50-mL plastic tubes. After centrifugation at400g for 30 min (400g at the bottom of tubes), a tumor cell-rich fractionwas collected from the 75% interface. The tumor-cell-enriched suspen-sion was then layered onto discontinuous gradients containing 4 mL eachof 25, 15, and 10% Percoll (Pharmacia) in complete medium in 15-n&plastic tubes. Centrifugation was performed at 25g for 7 min (25g at the


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144 SupinoTumor twiues

I enzymatic dIgestionsuspended In medium

. .

&100% Rcoll-Hypaque

1 4009 30 mm

Tumor cell-rich fraction

I 00g 30 mmTumor cell-rich fraction

Percoll

Tumor ce lls (purity more than 90%)

Fig. 4. Technique for Ficoll-Hypaque tumor cell purification from normalcells.

bottom of tubes), and tumor cells depleted of lymphoid cells were col-lected from the bottom and the 25% interface, washed, and suspended ncomplete medium at a concentration of 1 x 106/nL. The cells thus pre-pared were mainly tumor cells, with ~10% contamination by nonmalig-nant cells, as udged by morphological examination using Papanicolaoustaining or carcinoembryonic antigen staining for CEA-positive tumorcells. The cells were found to be more than 90-95% viable by the Trypanblue dye exclusion test. The mean yield of purified tumor cells was 2.3 x106, and the tumor cell count at the beginning of preparation was 13 xlo6 (rate of yield = 17.7).


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MTT Assays 1453.0 T /Al A--A--A----lr---.-A/AA

A--A-a --a------v~ -----A

o~--o-o/---o

Hours

Fig. 5. Effect of MTT concentration and incubation time on MTT reductionoptical den&y. 0 MTT 0.5 mA4and A MTT 1 mM on Ml9 melanoma cells; 0MTT 0.5 mM and A MTT 1 mM on SNB56 cells.4. Notes

1. Considerable changes in optical density are brought about by the presenceof different volumes of reaction medium. Low and constant evaporationmicrowell plates therefore have to be used (7), and variations over amedium volume htgher than 20 pL are unacceptable.2. Phenol red at 10 mg/mL does not change the optical density of formazan inDMSO.3. The MTT reductron is dependent on the o-glucose concentration in theculture medium and is independent of pH (8).4. MTT formazan production is dependent on the MTT concentration in theculture medium (9) (Fig. 5).5. The kinetics of MTT formazan production and the degree of saturationvary in a cell-line-specific manner.6. Drug cytotoxrcrty evaluation may be influenced by the length of exposureto MTT (9) (Fig. 6).7. Since MTT is cleaved by active mitochondria, the assay s effective, but to

a lesser extent, also in the absence of cell proliferation.8. For any given tumor cell line, the optical density of the solubilizedformazan product is directly proportional, over a wide range, to the num-ber of cells per well.


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146 Spin0

01 L -+--y--+- ----+ .-- - ,-. -- --A0 1 2 3 4 5

Hours

Fig. 6. Dependence of adrtamycin ID,, from MIT-time and concentrationexposure. 0 MIT 0.5 mA4and A MIT 1 mA4on Ml9 melanoma cells; 0 MIT0.5 n-H and A MTT 1 mA4 on SNB56 cells.

013E-1 1 10 100 1000Adrlamyan ( rig/m l )

Fig. 7. Dose-effect curves of adriamycin on N592 (0) and N592DX (0)cell lines.9. The MTT assay is effective on parental cell lines and their pleiotropicdrug-resistant counterpart, thus leading to a correlation between compa-rable cell lines (Fig. 7).10. The assay is effective on many different clmmal anticancer drugs(I) (Table 1).


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MTT Assays 147Table 1

CellsChemosensitivity of Cells Based on ICscs @tg/mL)of Some Clinical Anticancer Drugs in the MTT Reduction Assay

Dactinomycin Doxorubicin Ara-C Bleomycin CisplatinB16FlOB16B16FlL929Mouse spleenHPBMCLOVOK562RPM17272COL0320HSRL1210Clone AWiDrCOLO205MCF7COLO201

l>1>lO>lO>lO9.50>lO>lO>lO>lO>lO>lOCells Cycloheximide 5-FU Mitomycin Methotrexate VincristineB16FlOB16B16FlL929Mouse spleenHPBMCLOVOK562RPM17272COL0320HSRL1210Clone AWiDrCOLO205MCF7COLO20 1

0.04 0.01 0.008lO 9.20


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MTT Assays 1497. Twentyman, P. R. and Luscombe, M. (1987) A study of some variables in a tetra-zolium dye (MTT) based assay for cell growth and chemosensitivity. Br. J. Cancer

56,279-285.8. Plumb, J. A., Milroy, R., and Kaye, S. B. (1989) Effects of the pH dependence of3-(4,5-d~methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-formazan absorp-tion on chemosensitivity determined by a novel tetrazolium-based assay. CancerRes. 49,4435-4440.9. Vistica, D T , Skehan, P., Scudiero, D., Monks, A., Pittman, A., and Boyd, M. R.(1991) Tetrazolium-based assays for cellular viability: a critical examination ofselected paramenters affecting formazan production. Cancer Rex 51,25 15-2520.10. Jabbar, S. A. B., Twentyman, P. R., and Watson, J. V. (1989) The MIT assay under-estimates the growth inhibitory effects of interferons. Br. J. Cancer 60,523-528.

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