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The MTT Assay to Evaluate Chemosensitivity

Jack D. Burton

SummaryThe assessment of the degree or rate of cellular proliferation and cell viability is critical to the

assessment of the effects of drugs, antibodies, or cytokines on both normal and malignant cellpopulations. This can be accomplished by either direct or indirect counting methods. Directcounting by manual or automated methods, using a hemacytometer or particle counter, respec-tively, allows for serial cell counting at multiple time points, but these are low-throughputapproaches. High-throughput and robust alternatives to direct counting utilize either radiotracers(e.g., 3H-thymidine) or dye compounds, which can be adapted to multiwell culture plate for-mats. This chapter focuses on the use of tetrazolium-type indicator dyes, of which the compound3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) has been the most widelyutilized. Newer tetrazolium dyes that yield water-soluble products and offer added flexibility,increases in sensitivity, and fewer steps, which are offset by increased costs, are also covered.

Key WordsTetrazolium dye; MTT, proliferation; optical density; 50% inhibitory concentration; test

agent/drug.

1. IntroductionAssessment of the effects of drugs, antibodies, and cytokines on the prolif-

eration and viability of specific cell types grown in culture is a critical initialstep toward understanding and quantifying the effects of such agents. The abil-ity to evaluate the effects of these test agents in vitro allows for the screeningof a larger number of agents to identify those with the desired activity. In thecase of anticancer agents such as chemotherapy or other small-molecule drugs,those with the greatest antiproliferative activity are typically selected for invivo testing in appropriate animal models of human cancer. This is also truefor antitumor antibodies that are being evaluated as anticancer agents. Forimmunological studies involving either cytokines or antibodies, agonist or

From: Methods in Molecular Medicine, vol. 110: Chemosensitivity: Vol. 1: In Vitro AssaysEdited by: R. D. Blumenthal © Humana Press Inc., Totowa, NJ

growth-promoting effects are usually of interest. In this case, normal immunecell populations or factor-dependent cells lines are used to assess such effects.In all of these cases, the effect of these agents on total, viable cell numbers atthe end of the assay period is one of the key endpoints. Tetrazolium dyes havebeen shown to be sensitive, accurate, and efficient in the in vitro evaluation ofanticancer or immunological agents prior to preclinical and, ultimately, clini-cal testing.

3-(4,5-Dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) is ayellow, water-soluble tetrazolium dye, which crosses both plasma and mito-chondrial membranes. In the latter organelle of viable and glycolytically activecells, MTT is reduced by the action of NADH- or NADPH-dependent dehy-drogenases. Cellular reduction of MTT produces an insoluble, purple formazan.The amount of the formazan product that results from this reaction is dependenton the number of cells and their viability. As this reaction goes to completion,residual viable cells die, the formazan product can be solubilized, and the opti-cal density (OD) can be measured, which reflects the number of cells presentat the end of the assay period. This approach to identifying viable cells as wellas estimating cellular metabolic activity was first described using another tetra-zolium dye, 2,3,4-triphenyl tetrazolium chloride (TTC) (1–3). It was used to aidmicroscopic visualization of viable cells grown in soft agar in experimentsdesigned to assess in vitro growth conditions and cellular responses to drugtreatment (2,3). A quantitative approach to determining the metabolic activity oftracheal explants was developed by adding TTC to these cultures, dissolving theresulting formazan precipitates in an organic solvent, followed by determiningOD of the solubilized formazan product (1).

Mosmann (4) first described the design and setup of an assay using the MTTdye, in which cells are added to replicate wells of 96-well plates. The plates areplaced in an incubator for a sufficient time interval (usually 3–7 d) to allowcells to undergo several cell division cycles. At the end of the incubation period,MTT is added to each well followed by an additional incubation period of atleast 4 h to allow for complete bioreduction of MTT to the final formazanproduct. The OD of each well is determined in a plate reader, allowing a high-throughput, accurate, and sensitive method to estimate cell numbers. The orig-inal work with this dye was with growth factor–dependent cell lines, but it waslater shown to be useful for assessing growth characteristics and responses tochemotherapy drugs of cancer cell lines (5–7). Two groups of researchersaddressed the factors that affect the performance of MTT-based assays (6,7). Asdescribed in more detail below, the limitation of MTT and other earlier tetra-zolium dyes is the water insolubility of the formazan product. This led to thedevelopment and evaluation of other tetrazolium dyes whose formazan biore-

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duction products are water soluble. The first of this class of tetrazolium dyes tobe described was 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) (8,9). It was noted that with XTT (and later with othermembers of this class) it was necessary to add an electron-coupling reagent(typically phenazine methosulfate [PMS]) to achieve optimal absorbance values.When XTT/PMS was compared with MTT, similar absorbance values wereobtained with both methods, but the former method did not require either thecentrifugation or solubilization steps of the MTT method (8). Although XTT hasthis advantage, it has limited solubility and requires prewarming of the mediumto at least 37°C; it is also necessary to prepare XTT/PMS fresh just prior toadding it to the culture plate. Thus, other dyes in this class were synthesized andevaluated. The dye 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) was the next one to be described(10,11). As with XTT, PMS needs to be added to the MTS solution prior to itsaddition to cells. Unlike XTT, MTS solutions are stable, as are PMS solutions;thus, individual stock solutions of each can be stored and mixed just prior toadding to cells. MTS is available in a kit format containing stock solutions of both MTS and PMS from Promega. (Madison, WI). Another dye with improved characteristics over XTT has also been described, (4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1). It has beenshown to perform at least as well as MTT and XTT (12–15). Like MTS,WST-1 is stable in solution. It also requires the use of PMS. This dye is avail-able in kit format from Roche Diagnostics, GmbH (Penzberg, Germany). XTT,MTS, and WST-1 are all considerably more expensive than MTT, but the time that is saved, as well as the increased sensitivity and utility of these dyes with a wider range of cell lines, often makes them a fairly cost-effectivealternative.

2. Materials1. Sterile laminar flow hood or biosafety cabinet.2. Incubator (usually kept at 37°C with an internal atmosphere of 95% air/5% CO2).3. Cell culture medium and required additives (see Note 1).4. Phosphate-buffered saline (PBS) (see Note 2).5. Trypsin/EDTA solution.6. Sterile disposables including pipets, pipet tips, and tissue culture flasks.7. Repeating Eppendorf-type pipettor and compatible, sterile Combi-Tips (also help-

ful is an electronic pipettor with repetitive dispensing capability).8. 96-Well plate reader with adjustable, visible wavelength settings.9. Source of cells (usually continuously growing cell lines) (see Note 3).

10. Chemotherapeutic drugs (see Note 4).11. MTT.

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3. Methods3.1. Design of MTT Assays

The MTT assay is useful for measuring the effect of a wide range of com-pounds on the in vitro growth of either normal or cancer cell lines. The assayis set up in a 96-well, flat-bottomed polystyrene microtiter plate.

Cells are suspended in appropriate growth medium, and the cells are addedto replicate wells (triplicates are usually preferred) (see Note 5). It is preferableto add the cells to the required number of wells in the plate prior to adding thedrugs or agents to be tested. After the cells are added to the plate, it can beplaced on the incubator to allow cells to settle and attach (in the case of adher-ent cells) while the agents to be tested are being prepared.

Drugs or other compounds are added at defined concentrations to each set ofreplicate wells (see Notes 6 and 7). Agents to be tested must be properly solu-bilized. Aqueous solubility does not pose a problem for protein agents such asantibodies and cytokines. Many small-molecule compounds, however, have lim-ited water solubility. Most of these compounds can be dissolved in dimethylsulfoxide (DMSO)– or dimethyl formamide (DMF)–based solvents. DMSO ispreferable over DMF, because it tends to have slightly less toxicity for cellsin culture. Since most compounds of interest demonstrate antiproliferativeactivity at or below the micromolar level, they should be dissolved at concen-trations of 10–100 mM. This should result in a stock solution that has a con-centration of 1000–5000 times the highest concentration that will be tested inthe assay. This results in final concentrations of DMSO or DMF that usuallyexhibit minimal cellular toxicity. It is critical, however, to prepare control solu-tions of the identical dilutions of the solvent used for the stock solution foreach cell line to rule out or control for any solvent effects.

The choice of the concentration range to be tested depends on what is knownabout the agent in question and the cell line(s) to be tested. If little is knownabout the agent to be tested, a high initial concentration should be selected fol-lowed by approximately five serial dilutions to cover a range of at least 100-fold. To accomplish this, serial dilutions of threefold or higher are needed. Theprocedure for the preparation of serial dilutions is shown in Fig. 1. Once initialexperiments define the boundaries of the dynamic range of antiproliferativeactivity for a compound, a narrower concentration range should be evaluated.This can be accomplished by using approximately twofold dilutions. For aseries of twofold dilutions, the highest concentration is selected and prepared ina volume of at least 2 mL of the appropriate tissue culture medium (highervolumes are needed if a large number of plates are being set up). If 2 mL isselected for the final volume of the first tube, then 1 mL of medium is addedto each of the subsequent tubes in the series of dilutions. After the first tube is

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prepared by adding the correct volume of test agent from its stock solution fol-lowed by thorough mixing, 1 mL from the first tube is added to the secondtube. Tube 2 is then mixed, and 1 mL from this tube is added to tube 3 and soon to reach the required number of tubes (dilutions).

Cells are allowed to grow under incubator conditions (usually 37°C withsupplemental CO2). The planned duration of the assay determines the appro-priate number of cells to add to each well. For the assessment of the antipro-liferative effects of a wide range of compounds, an incubation period of 5–7 dis reasonable (see Note 8).

To maximize the dynamic range of this assay, cells that are untreated, whichserve as controls, should be allowed to proliferate until the level of cellularconfluence (the estimated percentage of the total area of the well that is coveredby cells) is 70–90%. This parameter should be monitored by inspecting platesdaily using an inverted microscope (see Note 9).

3.2. Processing of Plates for Adherent Cells

Once cells in the untreated wells have reached a confluency of 70–90%,5 mg/mL MTT is added to the plate (see Note 10). The approach to addingMTT and harvesting plates depends upon whether the cell lines or populationsare adherent or nonadherent in their growth patterns. While adherence to theplastic surface of microplate wells varies among cell lines, the line of demar-cation with respect to these assays is whether trypsin treatment is needed torelease cells from the surface of culture flasks. For such adherent cell types(usually epithelial and fibroblast-like cells), the processing procedure may becarried out as outlined next.

1. Prepare a stock solution of MTT in PBS. This final part of the assay does notrequire sterile conditions.

2. Remove the microplate from the incubator, and invert it over a container withmild shaking to remove most of the culture medium. While the plate is stillinverted, remove the remaining medium by blotting it on a small stack of papertowels.

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Fig. 1. Procedure for preparation of serial dilutions.

3. Prepare a working solution of MTT by diluting the stock solution 110 (finalconcentration: 0.5 mg/mL) using tissue culture medium (RPMI-1640-basedmedium should be used). Using a repeating pipettor, add 100 µL of this workingsolution to each well.

4. Cover the plate and place back in the incubator for at least 4 h to allow full con-version of the MTT.

5. After incubation, centrifuge the plate to pellet the precipitated formazan dye. Toaccomplish this, a standard tabletop centrifuge with an appropriate microplatecarrier is needed. Centrifuge the plates at 1000g for 10 min at ambient tempera-ture, and then invert the plate and blot onto paper towels to remove the bulk of theliquid.

6. Solubilize the MTT product (formazan) by adding 100 µL of DMSO to each well.To speed the rate of solubilization of the dye, the plate can be returned to the 37°Cincubator, with gentle tapping of the plate every 5–10 min. Solubilization is usuallycomplete within 30 minutes. An alternative approach to solubilize the MTT prod-uct in plates containing adherent cell types is to use an acid-isopropanol solvent asfollows. At the end of the initial culture period, the plate is inverted and blotted toremove the culture medium. In this case, the MTT working solution should be pre-pared using RPMI-1640 medium without added fetal bovine serum (FBS) at a110 dilution as described in step 3 and added to the plate at 100 µL/well. Afterthe requisite 4-h (or more) incubation period, the MTT product is solubilized by thedirect addition of an equal volume (in this case, 100 µL) of 0.04 N HCl in iso-propanol to each well. Since residual protein may precipitate if the resulting solu-tion is allowed to stand more than 30 min, plates should be read 5–15 min afteraddition of the acid-isopropanol.

3.3. Processing of Plates for Nonadherent Cells

Nonadherent cells cover the gamut of both normal and malignant cell linesof hematopoietic origin. While these cells settle and distribute fairly evenlyover the surface of the plates, changing medium in plates containing nonad-herent cells requires either removing the medium with a multichannel pipettoror centrifuging the plates followed by inverting and blotting them to remove themedium. The latter approach is much quicker. To facilitate the process, a mod-ification of the MTT method described in Subheading 3.2. is useful. (Thismethod may also be used with adherent cells.)

1. Dilute the same MTT stock solution (5 mg/mL) with an equal volume of tissueculture medium, and add 25 µL of this solution directly to each well with arepeating pipettor.

2. As with the adherent-cell method, return the plates to the incubator for a period ofat least 4 h.

3. Centrifuge the plates at 1000g for 10 min at ambient temperature, followed byinversion of the plates and blotting of excess medium.

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4. Use DMSO to solubilize the MTT formazan product as described in Subheading3.2., step 6.

3.4. Obtaining of OD Values

A standard microplate reader with adjustable wavelengths across the visiblespectrum is required. The detection wavelength for MTT should be between560 and 570 nm (see Note 11).

The OD values obtained for each set of triplicates corresponding to a specificconcentration of a test agent can then be transferred into a spreadsheet pro-gram such as Excel as described below.

3.5. Quantification and Calculation of Inhibitory Activity

The antiproliferative activity of agents is expressed as the 50% inhibitoryconcentration (IC50). This may be expressed as a molar concentration or on amass-per-volume basis such as micrograms per milliliter or milligrams per liter.The former mode of expression is preferable; it can be readily calculated usingthe molecular mass of the agent in question. Thus, the IC50 is defined as theconcentration of the test agent that results in a 50% decrease in the baselineor control level of proliferation. Since the readout of the MTT assay is OD, thebaseline for calculation of IC50 values is the OD value of the correspondingcontrol wells in the MTT assay plate. To perform this analysis, the appropriatecontrol OD value is essential. This control should be exactly matched for thedilution of the solvent that was used to prepare the stock solution of the testagent.

Using the control OD values, the percent inhibition at each concentration ofthe test agent can be calculated by dividing the observed OD value by the cor-responding control and multiplying by 100 as shown in the following equation:

1 – (ODobserved / ODcontrol) × 100 = % inhibition

Once the percent inhibition values corresponding to each concentration of thetest agent are calculated, these values can be plotted on a graph to allow forthe interpolation of the IC50 value. To obtain a linear or nearly linear plot, thepercent inhibition values should be log-transformed to obtain a linear equation,y = ax. The r2 value for the plot will indicate the goodness of fit for this equation.

4. Notes1. Cell culture medium is usually supplied according to specific, published formu-

lations, such as RPMI-1640 or Dulbecco’s modified Eagle’s medium. Such widelyused and published media formulations can usually be obtained from any estab-lished vendor.

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2. Vendors also supply standard stock solutions in sterile cell culture–tested formula-tions such as PBS (note that calcium/magnesium-free PBS is usually preferred)and trypsin/EDTA (needed to detach adherent cells). The latter two solutions aswell as other additives are supplied in concentrated stock solutions that either arediluted in sterile deionized water (in the case of PBS concentrates) or sterile PBS(in the case of trypsin/EDTA concentrates), or are added directly to media toachieve the desired final concentrations (in the case of antibiotics and L-glutamine).For any solutions that require dilution in deionized water, a source of highly purewater from which contaminating ions and volatile organics (activated charcoaladsorbed and exhibiting 18 mΩ of resistance) have been sufficiently removed iscritical.

3. Cell lines can be either obtained from individual investigators or purchased fromrepositories such as the American Type Culture Collection (ATCC; Manassas,VA). The published requirements of the cell lines (provided by the investigatorwho established the cell line or by the repository [e.g, ATCC]) will determine theadditives that are needed for the culture medium. Typically, basic cell culturemedium needs to be supplemented with FBS; antibiotics; L-glutamine; and, poten-tially, other additives. The specific preferred vendor from which to purchase thesecomponents is specified by the investigator or the repository such as ATCC.

4. For drugs that are not water soluble, organic solvents are required to prepare stocksolutions. DMSO is the most useful and least toxic of these solvents. Other alter-natives are DMF and ethanol. These should be obtained either in tissue cul-ture–tested formulations or in their most pure forms from any established supplierof fine chemicals. Although microbial contamination is unusual in organic sol-vents, it is still important that they be sterile filtered. To achieve an acceptablelevel of sterility, solutions must be filtered through filters with a cutoff of 0.45 µor less. For these organic solvents, a solvent-resistant syringe-tip filter is pre-ferred. For filtration of other aqueous solutions, syringe-tip or vacuum-assistedfilters may be used, depending on the volume of the solution that needs sterile fil-tration. Obtain information about the stability of the test agent or drug. If there isa question, prepare a new stock solution of it. Be sure to control as much as pos-sible for the effects of the organic solvents used to prepare drug stock solutions onthe growth of the cells by setting up control wells containing equal dilutions ofsolvent without the test agent or drug.

5. Cell lines should be maintained in culture medium to which they are adapted,and that supports consistent cellular proliferation. They should have maximumviability (>90% at the start of the assay) and be in the logarithmic phase of growthat the time of harvest. For most cell lines, this is either a basal or an enrichedmedium (e.g., RPMI-1640, Dulbecco’s modified Eagle’s medium, or Iscove’smodified Eagle’s medium) that is supplemented with bovine or other serum orpurified serum components as well as antibiotics and L-glutamine. The culturemedium in which cell line(s) are routinely carried should be used for setting upthe MTT assay.

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6. The final volume of medium in each well should be 200 µL. It is common toassess binary combinations of agents to examine the interaction of the two agents,which may be additive, synergistic, or antagonistic. In most instances, no morethan two agents need to be added to each well. Thus, it is convenient to first platethe cells in a volume of 100 µL of culture medium. This allows for a single agentto be added to each of the wells in a volume of 100 µL or, for two agents, in avolume of 50 µL for each agent.

7. To increase precision and reduce the setup time for assays, it is very helpful to uti-lize repeating pipettors. Commonly used repeating pipettors are those that aresimilar in design to one of the original instruments of this type, the Eppendorfrepeater. This pipettor requires specialized tips, which are often referred to asCombi-Tips. These must be sterile and are usually available in individually pack-aged and sterile form. These tips may also be used to add the agents to be testedto the individual wells, but it is more economical over time to use an electronicpipettor with a repeat pipetting mode. A 1000-µL size of such a pipettor willallow for the precise delivery of up to 20 separate 50-µL vol with a single stan-dard pipet tip. A standard manual pipettor may also be used.

8. For plates being incubated for 5 d or longer, an incubator chamber that is humid-ified should be used to minimize evaporative effects. To reduce the tendencytoward evaporation in the wells on the perimeter of the plate, the outer columnsand rows should not be used. To further reduce evaporation of the inner wells,200–250 µL/well of sterile water or PBS should be added to the outer perimeterwells.

9. For most cell lines, a total of 3–9 × 104 cells/well will result in the target level ofconfluence of 70–90%. To reach the target number of cells within the plannedtime frame (5–7 d), the doubling time of the cell line(s) needs to be estimatedfrom their current growth and maintenance characteristics. Typical doubling timesfor most mammalian cell lines range from 20 to 60 h. Thus, the number of cellsthat typically needs to be added to the wells of the plate at the start of the assayranges from 1.5 to 7.5 × 103/well. Estimating an appropriate cell number for ini-tial plating often requires repeat experiments to calibrate this parameter.

10. Observe the plate under an inverted microscope on a daily basis to assessthe level of confluence and the color of the medium. If the medium has taken ona yellowish color, it is depleted, and the MTT (or other dye) should be addedsoon.

11. If it is not possible to achieve maximum control OD values of >0.9 with MTT, trysubstituting one of the dyes that yields water-soluble end product (formazan),such as MTS or WST-1.

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