What is Dr. Zubair Khalid's research focus?

Dr. Zubair Khalid specializes in molecular virology, mRNA vaccine development, and computational biology, with a focus on avian pathogens like IBDV and Avian Reovirus.

Where is Dr. Zubair Khalid currently working?

Dr. Zubair Khalid is a Postdoctoral Research Associate at the University of Maryland (UMD), specifically within the Department of Animal and Avian Sciences.

MTT Assay for Cell Viability: Principles and Protocol

By Dr. Zubair Khalid, DVM, MS, PhD · 2026-08-11

Introduction to the MTT Assay for Cell Viability

The MTT assay is a colorimetric method for measuring cell viability, proliferation, and cytotoxicity based on the metabolic reduction of the yellow tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to an insoluble purple formazan product. Developed by Tim Mosmann in 1983 as a rapid alternative to radioactive thymidine incorporation assays, the MTT assay has become one of the most widely used endpoints in cell biology, drug screening, and toxicology.

The assay's utility rests on a simple principle: only metabolically active cells can reduce MTT, and the amount of formazan produced is proportional to the number of viable cells. This relationship enables quantitative comparisons of cell survival under different experimental conditions, including exposure to cytotoxic compounds, growth factor stimulation, or genetic manipulation. The assay is applicable to both adherent and suspension cell lines, requires no specialized equipment beyond a standard microplate reader, and can be completed within a few hours.

However, the apparent simplicity of the MTT assay belies a number of mechanistic nuances and technical variables that can compromise data quality if not properly controlled. Understanding the biochemistry of MTT reduction, the kinetics of formazan formation, and the factors that influence assay performance is essential for generating reproducible, biologically meaningful results. This article provides a comprehensive treatment of the MTT assay, from its mechanistic basis through protocol optimization and troubleshooting, with the goal of enabling rigorous experimental design and interpretation.

Mechanism of MTT Reduction

Role of Mitochondrial Dehydrogenases

MTT is a positively charged tetrazolium salt that readily penetrates viable cells. Once inside the cell, MTT is reduced to formazan, a deeply colored, water-insoluble crystal. The reduction reaction requires the transfer of electrons from reducing equivalents—primarily NADH and NADPH—to the tetrazolium ring, which is cleaved to yield the formazan product. This reaction is catalyzed by a class of enzymes known as oxidoreductases, with the mitochondrial succinate dehydrogenase complex historically considered the primary site of MTT reduction.

Succinate dehydrogenase (SDH), also known as mitochondrial complex II, catalyzes the oxidation of succinate to fumarate in the tricarboxylic acid (TCA) cycle, transferring electrons to ubiquinone. In the context of the MTT assay, SDH can transfer electrons from succinate to MTT, resulting in formazan formation. However, subsequent studies have demonstrated that MTT reduction is not exclusively mitochondrial. NAD(P)H-dependent oxidoreductases and dehydrogenases located in the cytosol, endoplasmic reticulum, and plasma membrane also contribute to MTT reduction. Indeed, the reducing capacity of a cell reflects its overall metabolic activity, not merely mitochondrial function. This is an important distinction: the MTT assay measures cellular metabolic activity as a proxy for viability, rather than directly quantifying cell number or membrane integrity.

The reduction of MTT by NADH and NADPH is non-enzymatic in principle, but the rate of reduction is greatly accelerated by enzyme-mediated electron transfer. The relative contribution of different enzyme systems varies by cell type, growth phase, and culture conditions. For example, proliferating cells typically exhibit higher rates of glycolysis and pentose phosphate pathway activity, leading to increased NADPH production and enhanced MTT reduction. This metabolic dependence means that the MTT signal reflects a snapshot of cellular redox state at the time of assay, which can be influenced by factors unrelated to cell number, as discussed below.

Factors Affecting Reduction

Several factors can influence the rate and extent of MTT reduction independent of cell viability:

Glucose concentration in the culture medium. The availability of glucose affects NADPH generation through the pentose phosphate pathway. High-glucose media generally support higher rates of MTT reduction than glucose-deprived conditions, even when cell numbers are identical.

Serum components. Fetal bovine serum (FBS) contains dehydrogenases and reducing agents that can directly reduce MTT, contributing to background signal. Serum-free conditions or reduced serum concentrations during the assay incubation period can minimize this effect.

pH and buffer composition. MTT reduction is pH-dependent, with optimal activity typically observed in the pH range of 6.5–7.5. Phenol red, a common pH indicator in culture media, does not interfere with absorbance readings at the formazan measurement wavelength (570 nm), but extreme pH shifts can affect enzyme activity and cell viability.

Cell density and confluency. At high cell densities, nutrient depletion and waste accumulation can reduce metabolic activity per cell, leading to a non-linear relationship between cell number and formazan production. This is a critical consideration for assay validation.

Time in culture. Cells in logarithmic growth phase exhibit higher metabolic activity than quiescent or senescent cells. Assays performed on freshly seeded versus long-term cultured cells may yield different absolute absorbance values for the same viable cell number.

Test compound interference. Many drugs and experimental compounds can directly reduce MTT or inhibit cellular reductases without affecting viability. This is a major source of false-positive or false-negative results and is addressed in detail in the troubleshooting section.

MTT Assay Protocol Step-by-Step

The following protocol describes a standard MTT assay for adherent cells in a 96-well plate format. Volumes and concentrations can be scaled for other formats, but the principles remain the same.

Cell Preparation and Seeding

Harvest cells from a subconfluent culture (70–80% confluency) using standard trypsinization procedures. For suspension cells, collect by centrifugation at 200 × g for 5 minutes. Ensure that cells are in logarithmic growth phase, as stationary-phase cells exhibit reduced metabolic activity.

Count cells using a hemocytometer or automated cell counter. Assess viability using trypan blue exclusion; the cell suspension should be >90% viable before seeding.

Prepare a cell suspension at the appropriate density in complete culture medium. The optimal seeding density depends on cell type and proliferation rate and must be determined empirically (see Section 5). For most adherent cell lines, seeding densities between 5,000 and 20,000 cells per well in a 96-well plate are appropriate for assays conducted 24–72 hours after seeding.

Seed the plate by adding 100 µL of cell suspension to each well. Include wells containing medium only (no cells) as blank controls. For experiments involving drug treatment, include vehicle controls (cells treated with the solvent used to dissolve the test compound) to account for any solvent effects.

Allow cells to attach by incubating at 37°C in a humidified 5% CO₂ atmosphere for 4–24 hours, depending on cell type. Primary cells may require longer attachment times; refer to Primary Cell Culture Guidelines for specific recommendations. Inspect the plate under an inverted microscope to confirm uniform attachment and distribution before proceeding.

Treat cells with test compounds at the desired concentrations and incubate for the appropriate exposure period. Include a positive control for cytotoxicity (e.g., 0.1% Triton X-100 or a known cytotoxic agent) to confirm assay sensitivity.

MTT Solution Preparation

Prepare MTT stock solution by dissolving MTT powder in phosphate-buffered saline (PBS) at a concentration of 5 mg/mL. MTT is light-sensitive and should be handled under reduced light conditions. Sterile-filter the solution through a 0.22 µm filter and store at 4°C protected from light. The stock solution is stable for several weeks, but fresh preparation is recommended for optimal results.

Dilute the stock solution in pre-warmed culture medium (without serum or phenol red, if possible) to a working concentration of 0.5–1.0 mg/mL. The final MTT concentration in the well should be 0.5 mg/mL when 10 µL of a 5 mg/mL stock is added to 100 µL of culture medium.

Incubation and Formazan Solubilization

Add MTT solution to each well. For a 96-well plate containing 100 µL of culture medium, add 10 µL of the 5 mg/mL MTT stock solution, yielding a final concentration of 0.45 mg/mL. Gently tap the plate to mix.

Incubate the plate at 37°C for 2–4 hours. The optimal incubation time depends on cell density and metabolic activity. Check the plate periodically under a microscope; the appearance of purple crystals within cells indicates that formazan is being produced. Incubation times longer than 4 hours are generally not recommended, as they can lead to reduced cell viability and non-linear signal.

Remove the medium carefully using a multichannel pipette or by inverting the plate and blotting on absorbent paper. Take care not to dislodge the formazan crystals or adherent cells. For suspension cells, centrifuge the plate at 400 × g for 5 minutes before removing the supernatant.

Add solubilization solution to dissolve the formazan crystals. The most commonly used solvent is dimethyl sulfoxide (DMSO), added at 100–150 µL per well. Alternatively, acidified isopropanol (0.04 N HCl in isopropanol) or a 10% sodium dodecyl sulfate (SDS) solution in 0.01 N HCl can be used. DMSO is generally preferred for its superior solubilizing capacity, but it is incompatible with some plasticware and can dissolve the plate itself if left for extended periods.

Mix thoroughly by pipetting up and down or using a plate shaker for 5–10 minutes. Ensure that all formazan crystals are completely dissolved. The solution should appear uniformly purple with no visible particulates.

Absorbance Measurement

Measure absorbance at 570 nm using a microplate reader. The reference wavelength should be 630–690 nm to subtract background absorbance from cell debris and plate artifacts. If a reference wavelength is not available, subtract the absorbance of blank wells (medium only) from all sample readings.

Read the plate within 30–60 minutes of adding the solubilization solution. Formazan in DMSO is stable for several hours, but prolonged exposure to light can cause photodegradation.

Record raw absorbance values for subsequent analysis. Ensure that the microplate reader is calibrated and that absorbance values fall within the linear range of the instrument (typically 0.1–2.0 optical density units).

Data Analysis and Interpretation

Calculating Viability Percentage

The raw absorbance values from the MTT assay are proportional to the number of metabolically active cells, but they must be normalized to appropriate controls to yield meaningful viability data. The standard calculation is:

Viability (%) = (Absorbance of treated sample − Absorbance of blank) / (Absorbance of untreated control − Absorbance of blank) × 100

Where:

Absorbance of treated sample is the raw reading from cells exposed to the test condition
Absorbance of untreated control is the reading from cells cultured under identical conditions without treatment
Absorbance of blank is the reading from wells containing medium and MTT but no cells

This calculation assumes a linear relationship between absorbance and viable cell number, which must be validated for each experimental system. A detailed explanation of the underlying calculations is provided in the Calculate Cell Viability resource.

For dose-response experiments, viability data are typically plotted against the logarithm of drug concentration, and the half-maximal inhibitory concentration (IC₅₀) is determined by non-linear regression using a four-parameter logistic model. Most statistical software packages (GraphPad Prism, R, etc.) include built-in functions for this analysis.

Statistical Considerations

The MTT assay is subject to well-to-well and plate-to-plate variability, necessitating appropriate experimental design and statistical analysis:

Technical replicates. Each condition should be tested in at least 3–6 replicate wells. Technical replicates account for pipetting errors and plate position effects.

Biological replicates. Independent experiments performed on different days with different cell passages are essential to confirm that observed effects are reproducible. A minimum of three independent biological replicates is standard practice.

Plate position effects. Cells in edge wells often experience differential evaporation and temperature gradients, leading to edge effects. Avoid using the outermost wells for experimental conditions, or fill them with PBS or medium to reduce evaporation.

Normalization. When comparing across experiments, normalize viability data to the untreated control within each plate. This controls for inter-experimental variation in absolute absorbance values due to differences in cell density, passage number, or reagent lot.

Statistical tests. For comparisons between two groups, use a two-tailed Student's t-test. For multiple comparisons, use one-way ANOVA followed by a post-hoc test (e.g., Dunnett's test for comparison to a single control). Non-parametric alternatives (Mann-Whitney U test, Kruskal-Wallis test) should be used when data are not normally distributed or when sample sizes are small.

Optimization of MTT Assay Conditions

The MTT assay requires optimization for each cell type and experimental context. The following parameters should be systematically evaluated before conducting large-scale experiments.

Determining Optimal Cell Number

The relationship between cell number and absorbance must be linear for the assay to be quantitative. To establish this relationship:

Seed a range of cell densities (e.g., 1,000 to 50,000 cells per well in a 96-well plate) in replicate wells.
Allow cells to attach overnight, then perform the MTT assay using standard conditions.
Plot absorbance against cell number. The linear range is the region where absorbance increases proportionally with cell number.

Select a seeding density within the middle of the linear range for subsequent experiments. This ensures that both increases (proliferation) and decreases (cytotoxicity) in cell number can be detected without saturating or falling below the detection limit. For most cell lines, the linear range spans approximately 1,000–20,000 cells per well, but this varies considerably. Highly metabolically active cells may saturate the signal at lower densities, while quiescent cells may require higher densities for detectable signal.

MTT Concentration and Incubation Time

The standard MTT concentration of 0.5 mg/mL with a 2–4 hour incubation is appropriate for most applications, but optimization may improve signal-to-noise ratio:

MTT concentration. Higher concentrations (up to 1 mg/mL) increase the rate of formazan production but can also increase background absorbance and may be toxic to cells during extended incubations. Lower concentrations (0.1–0.25 mg/mL) reduce background but require longer incubation times.

Incubation time. The optimal incubation time is the shortest period that produces a robust, reproducible signal. Perform a time-course experiment by measuring absorbance at 1, 2, 3, and 4 hours after MTT addition. Select the time point at which the signal is in the linear range and the untreated control absorbance is between 0.5 and 1.5 optical density units.

Kinetic considerations. The rate of formazan production decreases over time as MTT is depleted and as the culture medium becomes acidified by cellular metabolism. Extended incubations (>6 hours) can lead to cell death and reduced signal, particularly in serum-free conditions.

Solubilization Solvent Selection

The choice of solubilization solvent affects the efficiency of formazan dissolution and the stability of the final colored solution:

DMSO. The most widely used solvent, DMSO dissolves formazan rapidly and completely. It is compatible with most microplate readers and provides a stable signal for several hours. However, DMSO is hygroscopic and can absorb water from the atmosphere, leading to precipitation of formazan if plates are left uncovered. DMSO is also incompatible with polystyrene plates if left for extended periods (>30 minutes), as it can dissolve the plastic.

Acidified isopropanol. A solution of 0.04 N HCl in isopropanol is an effective alternative that avoids some of the compatibility issues of DMSO. The acid helps to dissolve formazan and also fixes cells, preventing further metabolic activity. However, acidified isopropanol can cause protein precipitation, which may increase background absorbance.

SDS-HCl solution. A 10% SDS solution in 0.01 N HCl is gentler on cells and can be used for assays where the cells are not removed before solubilization. This solvent is particularly useful for suspension cells, as it lyses cells and dissolves formazan in a single step.

Optimization approach. Test each solvent with a known number of cells and compare the absorbance values, background readings, and signal stability over time. Select the solvent that provides the highest signal-to-background ratio and the most stable readings.

Common Pitfalls and Troubleshooting

Low Formazan Solubility

Symptom: Absorbance readings are low and variable, or visible purple crystals remain in the well after solubilization.

Causes: Incomplete solubilization of formazan crystals, particularly when cells are dense or when the solubilization solution is added in insufficient volume. Formazan crystals can also become trapped within cell monolayers, especially in wells that were allowed to dry before solubilization.

Solutions: Increase the volume of solubilization solution to 150–200 µL per well. Extend the mixing time on a plate shaker to 10–15 minutes. For stubborn crystals, pipette the solution up and down vigorously. If using acidified isopropanol, ensure that the solution is freshly prepared, as aged solutions lose effectiveness. Consider switching to DMSO, which has superior solubilizing capacity.

Interference by Reducing Agents

Symptom: High absorbance in blank wells or in wells containing test compound but no cells.

Causes: Many compounds can directly reduce MTT to formazan in a cell-free manner. Common culprits include ascorbic acid, glutathione, dithiothreitol (DTT), β-mercaptoethanol, and certain plant-derived polyphenols. Additionally, some drugs and nanoparticles exhibit intrinsic absorbance at 570 nm, contributing to false signal.

Solutions: Include cell-free controls containing the test compound and MTT to assess direct reduction. If interference is detected, consider using a different viability assay, such as the ATP-based assay, which is less susceptible to reducing-agent interference. Alternatively, wash cells with fresh medium before adding MTT to remove residual test compound. For compounds with intrinsic absorbance at 570 nm, measure absorbance at a different wavelength or use a reference wavelength to subtract background.

Cell Loss During Washing

Symptom: Low and variable absorbance readings, particularly in treated wells.

Causes: The medium removal step before solubilization can dislodge loosely attached cells, especially if cells are dying or detaching due to treatment. This is a particular problem for cytotoxic compounds that cause cell rounding and detachment.

Solutions: Centrifuge the plate (for suspension cells) or handle the plate gently when removing medium. Use a multichannel pipette rather than inverting the plate. Alternatively, add the solubilization solution directly to the culture medium without removing it, provided that the solubilization solution is compatible with the medium components. Some protocols use a 10% SDS-HCl solution that can be added directly to the culture medium, lysing cells and dissolving formazan in a single step.

Non-Linear Relationship Between Cell Number and Absorbance

Symptom: Absorbance does not increase proportionally with cell number, or the signal saturates at high cell densities.

Causes: At high cell densities, nutrient depletion, oxygen limitation, and waste accumulation reduce metabolic activity per cell. Additionally, the MTT substrate may become limiting, or formazan crystals may interfere with light transmission.

Solutions: Reduce the seeding density to within the linear range. Shorten the MTT incubation time to reduce substrate depletion. Ensure that the culture medium is freshly prepared and that cells are in logarithmic growth phase at the time of assay.

High Background Absorbance

Symptom: Blank wells (medium only) show absorbance values above 0.1.

Causes: MTT can be reduced by components of the culture medium, particularly serum and certain reducing agents. Phenol red can also contribute to background if the measurement wavelength is not optimal.

Solutions: Use serum-free medium during the MTT incubation step. Use phenol red-free medium if available. Ensure that the blank wells contain the same volume of medium and MTT as the sample wells. Measure absorbance at 570 nm with a reference wavelength of 630–690 nm to subtract non-specific background.

Comparison with Other Viability Assays

Advantages of MTT

The MTT assay offers several advantages that have contributed to its widespread adoption:

Simplicity and cost-effectiveness. The assay requires only a tetrazolium salt, a solubilization solvent, and a microplate reader. No specialized equipment or radioactive reagents are needed.

Wide applicability. The assay works with both adherent and suspension cells, and with primary cells and established cell lines. It is compatible with standard 96-well plate formats, enabling high-throughput screening.

Quantitative output. The absorbance reading provides a continuous measure of metabolic activity, allowing for dose-response analysis and statistical comparison.

Established literature base. The MTT assay has been used in tens of thousands of published studies, providing a wealth of comparative data and established protocols for specific cell types.

Limitations and Alternatives

The MTT assay has several well-documented limitations that should be considered when selecting a viability assay:

Insoluble formazan product. The requirement for a solubilization step adds time and introduces a potential source of variability. The water-soluble tetrazolium salts—MTS, XTT, and WST-1—produce soluble formazan products that can be measured directly in the culture medium, eliminating the solubilization step. These assays are more convenient for kinetic studies and high-throughput applications.

Metabolic dependence. The MTT assay measures metabolic activity, not cell number or membrane integrity. Compounds that affect cellular metabolism without affecting viability (e.g., metabolic inhibitors, glucose deprivation) can produce false results. The Measure Cell Viability resource provides an overview of alternative approaches.

Reducing-agent interference. As discussed above, the MTT assay is susceptible to interference from compounds that directly reduce tetrazolium salts. The WST assays, particularly WST-1 and WST-8, are less sensitive to this interference because they require an intermediate electron carrier (e.g., 1-methoxy-5-methylphenazinium methyl sulfate) for reduction.

Cytotoxicity of MTT itself. MTT is mildly toxic to cells, and the formazan crystals can damage cellular membranes during the incubation period. This is generally not a problem for endpoint assays, but it precludes the use of MTT for longitudinal studies of the same cells.

ATP-based assays. The ATP assay measures cellular ATP content using the luciferase-luciferin reaction. This assay is more sensitive than MTT, has a wider linear range, and is less affected by metabolic state. However, it requires cell lysis and is more expensive per sample.

Comparison table:

Assay	Principle	Solubility of Product	Sensitivity	Interference by Reducers	Cost per Sample
MTT	Reduction to formazan	Insoluble (requires solubilization)	Moderate	High	Low
MTS	Reduction to formazan	Soluble	Moderate	High	Low
XTT	Reduction to formazan	Soluble	Moderate	High	Low
WST-1	Reduction to formazan	Soluble	Moderate-High	Low	Moderate
ATP	Luciferase reaction	N/A	High	Low	High

Applications of the MTT Assay

Drug Cytotoxicity Screening

The MTT assay is a cornerstone of preclinical drug development, providing a rapid and quantitative measure of the cytotoxic effects of candidate compounds. In a typical screening workflow:

Dose-response analysis. Cells are treated with a range of compound concentrations (typically 0.001–100 µM in half-log or log increments) for 24–72 hours. The resulting viability curve yields the IC₅₀, which quantifies the potency of the compound.

Combination studies. The MTT assay can be used to assess the synergistic, additive, or antagonistic effects of drug combinations. The combination index (CI) is calculated using the Chou-Talalay method, which requires dose-response data for each drug alone and in combination.

Structure-activity relationship (SAR) studies. By testing a series of structurally related compounds, the MTT assay enables the identification of functional groups that enhance or diminish cytotoxicity.

Resistance profiling. The MTT assay can compare the sensitivity of drug-resistant and drug-sensitive cell lines to identify mechanisms of resistance and to guide the development of second-line therapies.

When using the MTT assay for drug screening, it is essential to include appropriate controls: vehicle-treated cells, a known cytotoxic positive control, and cell-free wells containing the test compound to assess direct MTT reduction.

Cell Proliferation Studies

The MTT assay can also be used to measure cell proliferation, provided that the relationship between cell number and absorbance is linear and that the assay is performed during the logarithmic growth phase. Typical applications include:

Growth factor and cytokine stimulation. Cells are cultured in reduced-serum or serum-free medium to induce quiescence, then stimulated with the factor of interest. The MTT assay is performed at various time points to measure the proliferative response.

Conditioned medium testing. The effect of conditioned medium from one cell type on the proliferation of another cell type can be assessed using the MTT assay.

Genetic manipulation. The effect of gene overexpression, knockdown, or knockout on cell proliferation can be quantified by comparing MTT absorbance between modified and control cells over time.

Long-term proliferation assays. For experiments lasting several days, cells may need to be passaged to prevent overconfluency. Standard Cell Passaging procedures should be followed, and the MTT assay should be performed on a subset of wells at each time point.

For proliferation studies, it is important to note that the MTT assay measures the total metabolic activity of the cell population, which reflects both cell number and the proliferative state of individual cells. Cells in S phase exhibit higher metabolic activity than quiescent cells, so the MTT signal may overestimate the increase in cell number during active proliferation.

Summary and Best Practices

Key Takeaways

The MTT assay is a robust and versatile method for assessing cell viability, but its reliability depends on careful optimization and rigorous experimental design. The following principles are essential:

Validate linearity. Establish the linear relationship between cell number and absorbance for each cell type and experimental condition before interpreting results.

Control for interference. Include cell-free controls to detect direct MTT reduction by test compounds, and use appropriate blank and vehicle controls.

Optimize conditions. Determine the optimal cell density, MTT concentration, incubation time, and solubilization solvent for each experimental system.

Use appropriate statistical design. Include technical and biological replicates, and use appropriate statistical tests for data analysis.

Interpret with caution. The MTT assay measures metabolic activity as a surrogate for viability. Confirm key findings with an independent method, such as ATP measurement or clonogenic assay.

Checklist for Reproducibility

[ ] Cells are in logarithmic growth phase and >90% viable at the time of seeding
[ ] Seeding density is within the validated linear range
[ ] MTT stock solution is freshly prepared or stored appropriately (4°C, protected from light)
[ ] MTT working concentration and incubation time are optimized for the cell type
[ ] Blank wells (medium only) and vehicle controls are included on every plate
[ ] Cell-free controls for test compound interference are included
[ ] Solubilization is complete (no visible crystals) before absorbance measurement
[ ] Absorbance is measured at 570 nm with reference wavelength subtraction
[ ] Readings are taken within 60 minutes of solubilization
[ ] Data are normalized to untreated controls and analyzed with appropriate statistics
[ ] Key findings are confirmed with an independent viability assay

Frequently Asked Questions

What is the MTT cell viability assay?

The MTT assay is a colorimetric method for measuring cell viability and proliferation based on the reduction of the yellow tetrazolium salt MTT to purple formazan crystals by metabolically active cells. The amount of formazan produced, quantified by absorbance measurement, is proportional to the number of viable cells. It is widely used in drug screening, cytotoxicity testing, and cell biology research.

How does the MTT assay work?

MTT enters cells and is reduced by NAD(P)H-dependent oxidoreductases and dehydrogenases, including mitochondrial succinate dehydrogenase. This reduction cleaves the tetrazolium ring, producing an insoluble purple formazan crystal. The formazan is solubilized in an organic solvent (typically DMSO), and the absorbance is measured at 570 nm. Since only metabolically active cells can reduce MTT, the absorbance signal is directly proportional to the number of viable cells.

What is the protocol for MTT cell viability assay?

The basic protocol involves: (1) seeding cells in a 96-well plate and allowing attachment; (2) treating cells with test compounds for the desired exposure period; (3) adding MTT solution (final concentration 0.5 mg/mL) and incubating at 37°C for 2–4 hours; (4) removing the medium and adding a solubilization solution (e.g., DMSO) to dissolve formazan crystals; (5) measuring absorbance at 570 nm with a reference wavelength of 630–690 nm; and (6) calculating viability as a percentage of untreated controls. Detailed steps are provided in Section 3 of this article.

Why is my MTT assay not working?

Common reasons for MTT assay failure include: (1) insufficient cell density, resulting in low signal; (2) excessive cell density, causing signal saturation and non-linearity; (3) interference by test compounds that directly reduce MTT; (4) incomplete solubilization of formazan crystals; (5) cell loss during the medium removal step; and (6) use of aged or improperly stored MTT solutions. Systematic troubleshooting should address each of these variables, as described in Section 6.

Can I use the MTT assay for adherent cells?

Yes, the MTT assay is widely used for adherent cells. The key considerations are ensuring uniform cell attachment across wells, avoiding cell loss during the medium removal step, and ensuring complete solubilization of formazan crystals from the cell monolayer. For loosely adherent cells, consider centrifuging the plate before removing the medium or using a solubilization solution that can be added directly to the culture medium.

What is the difference between MTT and MTS assay?

The MTT and MTS assays both measure cellular metabolic activity through tetrazolium reduction, but they differ in the properties of their formazan products. MTT produces an insoluble formazan that requires a solubilization step, while MTS produces a water-soluble formazan that can be measured directly in the culture medium. The MTS assay is more convenient for kinetic studies and high-throughput applications, but it requires an intermediate electron carrier (phenazine methosulfate) for efficient reduction. MTT is generally more sensitive and less expensive, but the additional solubilization step introduces more variability.

How do I calculate cell viability from MTT assay results?

Cell viability is calculated by normalizing the absorbance of treated samples to untreated controls. The formula is: Viability (%) = (Absorbance of treated sample − Absorbance of blank) / (Absorbance of untreated control − Absorbance of blank) × 100. This calculation assumes a linear relationship between absorbance and viable cell number, which should be validated for each experimental system. For dose-response experiments, viability data are plotted against drug concentration to determine the IC₅₀.