The MTT assay is a reliable tool for evaluating cellular health and metabolic activity. It is essential in several research fields, such as drug discovery, cancer research, toxicology, and tissue engineering. It provides quantitative data essential for understanding cellular responses to drugs, toxins, and environmental stressors and is vital for studies on apoptosis, necrosis, and metabolic adaptations. However, optimizing experimental conditions is key to minimizing artifacts and ensuring reliable results.

Principle and mechanism of the MTT assay

The MTT assay relies on the capacity of live cells to convert the yellow MTT tetrazolium salt into purple formazan crystals^1,2. The change from yellow to purple gives a visual signal for cell viability.

Reduction of MTT is achieved by the action of mitochondrial dehydrogenases, eg, NAD(P)H-dependent oxidoreductases, which cleave the tetrazolium ring and reduce MTT to formazan. A central role is played by succinate dehydrogenase, which is implicated in electron transfer in the mitochondria. The efficiency of the reduction is directly correlated with mitochondrial integrity, and the assay thus serves as a trustworthy measure of cell viability.

Apart from mitochondria, MTT reduction also occurs in other cellular compartments, including the endoplasmic reticulum (ER), lysosomes, and plasma membrane³. Therefore, the assay reflects the overall metabolic activity and reduces the power of the cell.

Following the MTT reduction, the produced formazan is quantified using a spectrophotometer. The amount of formazan correlates with cell viability and metabolic activity, making the assay highly sensitive to cytotoxic agents. Any disruption of the enzymatic processes by toxic chemicals or environmental stressors leads to reduced formazan production, indicating decreased cell viability.

It is important to note that cellular stress can impact various reduction pathways differently, which may affect the interpretation of the results. Thus, MTT assay findings must consider the cellular context and the potential effects of stressors on different cellular processes.

Reagents and materials for the MTT assay

Our MTT assay kit ab211091 offers a convenient, non-radioactive, and high-throughput solution for assessing cell proliferation, viability, and cytotoxicity.

The MTT assay is based on a number of essential reagents that allow the measurement of cell viability and metabolic activity with accuracy. Each element has an important role to play in making the assay reliable and sensitive⁴.

MTT powder is the primary substrate used in the assay⁴. This yellow, water-soluble tetrazolium salt is reduced by mitochondrial enzymes in viable cells, forming purple formazan crystals. The stability and activity of MTT are essential for reliable results.

To maintain its integrity, MTT powder should be stored away from light and moisture. Exposure to light or moisture can degrade MTT, leading to increased background absorbance and reduced assay accuracy. Proper storage ensures that MTT retains its full activity, which is essential for obtaining sensitive and reproducible results.

PBS is used as the solvent for preparing the MTT stock solution, providing a physiological pH (~7.4) and isotonic conditions that are essential for maintaining MTT stability and supporting optimal reduction by cells⁴.

It is vital to use sterile PBS with the correct pH to preserve MTT’s activity. Non-sterile PBS may introduce contaminants that could interfere with the assay, while improper pH can negatively affect MTT solubility and stability. PBS should be prepared following standard protocols to ensure its isotonicity and pH, which are suitable for maintaining cell viability and ensuring efficient MTT reduction⁵.

MTT solubilization solutions

• Dimethyl sulfoxide (DMSO): DMSO is commonly used to dissolve formazan crystals generated in the MTT assay due to its excellent solubility properties. For optimal results, pure anhydrous DMSO should be used.
  However, caution is necessary as DMSO can be cytotoxic at higher concentrations and may adversely affect cell viability if not appropriately handled.
• Acidified isopropanol: Acidified isopropanol, typically containing around 0.04 N hydrochloric acid (HCl), is an effective alternative to DMSO for dissolving formazan crystals. The acidic environment enhances the solubility of formazan, stabilizing the product and making it a reliable choice for solubilization in the MTT assay.
• Sodium dodecyl sulfate (SDS) solution: 10% SDS solution in 10 mM HCl is used to dissolve formazan crystals in cell types that are resistant to other solvents. SDS aids in the lysis of cell membranes, enhancing formazan solubility. However, its use should be considered with caution, as SDS can interfere with subsequent biochemical analyses due to its disruptive effects on cellular components.

Serum-free medium is used during the MTT incubation step to prevent interference from serum proteins, which can skew the assay results. Serum components may either enhance or inhibit MTT reduction, leading to inaccurate measurements. Using serum-free medium ensures an environment free from these potential artifacts, allowing metabolic activity to more accurately reflect cell viability⁶.

Materials: The success of the MTT assay also relies on the quality and suitability of the materials utilized⁷.

96-well microplates are essential for both cell culture and absorbance measurements. The flat bottom ensures optimal cell adhesion and uniform light path lengths for accurate spectrophotometric readings. For adherent cells, tissue culture-treated plates are pivotal to promoting proper attachment and growth.

A microplate reader measures the absorbance of solubilized formazan, indicating cell viability. The optimal wavelength for measuring formazan is 570 nm, with a reference wavelength of 630 nm used to correct for background noise. It is essential to ensure the spectrophotometer is calibrated correctly before use, as inaccurate calibration can lead to erroneous absorbance readings, affecting the interpretation of cell viability data. The reader should be capable of measuring absorbance in the 500-600 nm range to accommodate different solvents and variations in formazan absorbance peaks.

An orbital shaker is utilized during formazan solubilization to facilitate the even mixing of the solvent and formazan crystals. Gentle agitation ensures the complete dissolution of the crystals, which is vital for precise absorbance measurements. Overshaking should be avoided, as it could cause splashing or cross-contamination between wells, resulting in inaccurate outcomes.

Vortex mixers or sonicators can be used to dissolve MTT powder when preparing the initial stock solution, especially if the powder clumps. Ensuring a uniform solution is essential for accurate assay results, as it guarantees consistent reduction efficiency across all wells, leading to reliable cell viability measurements.

MTT assay method

Optimization of cell seeding density:

Adjusting the best cell seeding density is important for obtaining precise results in the MTT assay. Suggested cell seeding densities may range from 1,000 to 100,000 cells per well on a 96-well plate, depending upon the cell line and the intention of the study⁴. For example, leukemic cell lines commonly need 0.5-1.0×10⁵ cells/ml, whereas cell lines of solid tumors might demand densities between 1×10⁴ to 1.5×10⁵ cells/ml.

Cells must be in logarithmic growth to provide maximum metabolic activity and increased sensitivity to therapy⁴. The cells are actively dividing during this growth phase, which will provide the correct response to experimental conditions.

Incorrect seeding density might jeopardize the assay. Low density will provide less formazan production, so detecting a distinct signal will be challenging. In contrast, high density might produce nutrient depletion, crowding of cells, and changing metabolic activity, so the results might be skewed.

Selection of plate format:

The 96-well microplate format is the most popular for the MTT assay, suitable for high-throughput screening. Other formats, such as 24-well or 6-well plates, can also be used, but reagent volumes and cell numbers have to be adjusted. Ensuring uniform cell density per unit of surface area in all wells is important to guarantee reproducibility and accurate data.

Maintaining aseptic environment:

Sterility is important to avoid contamination, which can produce false results. Microbial contamination can lead to false-positive signals through MTT metabolism, while nutrient competition by contaminants can produce false-negative signals.

Additionally, it is essential to handle sterile reagents and disposable consumables in a laminar flow hood to ensure aseptic environments and reduce the risk of contamination.

Protocol for adherent cells

After culturing and treating cells, the following protocol should be followed to ensure reproducible quantification of adherent cell viability, balancing metabolic activity detection with procedural precision^1,4,5,6.

• Aspirate the spent culture medium from each well using a pipette or vacuum system, ensuring adherent cells remain undisturbed. The bottom of the well should not be touched to avoid cell detachment.
• Add 50 µL of serum-free medium to each well to remove residual serum components that may interfere with MTT reduction. Swirl the plate gently for even coverage.
• Add 50 µL of MTT solution (5 mg/mL in PBS) to each well. Ensure the solution is filter-sterilized (0.22 µm) and stored at -20°C, protected from light to maintain stability.
• Incubate the plate at 37°C with 5% CO₂ for 2-4 hours. Monitor wells periodically for purple formazan crystal formation, indicating mitochondrial activity. Do not exceed 6 hours to prevent cell stress.
• Aspirate the MTT solution carefully without disturbing the formazan crystals. If necessary, centrifuge the plate at 300-500 × g for 5 min to pellet loosely adherent crystals.
• Add 100-150 µL of solubilization solvent (eg, DMSO, acidified isopropanol, or 10% SDS in 0.01 M HCl) to dissolve the formazan crystals.
• Wrap the plate in aluminum foil or keep it in darkness to prevent formazan degradation. Shake the plate gently (100-150 rpm) for 10-15 min, and pipette stubborn crystals to ensure uniform dissolution.
• Measure the absorbance at 570 nm with a 630 nm reference wavelength to correct for background noise. Complete readings within 1 hour to prevent crystal precipitation or solvent evaporation.

Protocol for suspension cells

The steps below provide the detailed MTT assay protocol using suspension cells^4,6:

• Centrifuge the microplate at 1,000 x g for 5 min at 4°C to pellet the suspension cells at the bottom of the wells.
• Aspirate the supernatant carefully without disturbing the cell pellet.
• Add 50 µL of serum-free medium to each well to remove any residual culture components that may interfere with MTT reduction.
• Add 50 µL of MTT solution (5 mg/mL in PBS) to each well. Viable cells reduce MTT to purple formazan, indicating metabolic activity.
• Resuspend the cell pellet gently in the MTT solution by pipetting up and down to ensure even distribution.
• Incubate the plate at 37°C with 5% CO₂ for 2-4 h to allow MTT reduction to formazan crystals.
• Centrifuge the microplate at 1,000 x g for 5 min at 4°C to pellet the cells with formazan crystals.
• Aspirate the supernatant carefully without disturbing the formazan pellet.
• Add 100-150 µL of MTT solvent (eg, DMSO or isopropanol) to dissolve the formazan crystals.
• Pipette the solution gently up and down to ensure complete solubilization of the formazan crystals.
• Measure absorbance at 570 nm (or 590 nm) using a spectrophotometer with a reference wavelength of 630 nm to correct background absorbance. The absorbance is proportional to the number of viable cells in each well.

Analyzing and interpreting MTT assay data

The MTT assay provides important information regarding cell viability and metabolic activity, but proper interpretation of the data is required for meaningful conclusions. This encompasses various steps, such as calculating cell viability percentages, plotting dose-response curves, and determining the implications of absorbance values.

Calculating cell viability percentages

To determine cell viability, proper control wells need to be included in the experimental design along with the treated wells⁷. You should include two types of control wells.

1. Blank wells: Wells filled with cell culture medium and the MTT reagent but not containing cells are known as blank wells. They are used to assess background absorbance from the medium or non-specific MTT reduction. Subtracting the absorbance of blank wells isolates the formazan signal, providing precise results.
2. Untreated control wells (negative control): These wells are filled with cells grown in regular conditions without any treatment. They depict basal metabolic activity and are deemed to be 100% viable.

The treated wells (experimental wells) contain the cells that have been treated with the test chemicals (drugs or compounds). The absorbance of these wells shows how the treatment affected the metabolic activity and cell viability.

By using the formula below, you can determine the cell viability⁸:

This equation standardizes the data by adjusting for background absorbance from blank wells.

Generating dose-response curves

Dose-response curves are essential for evaluating the effect of varying concentrations of a test agent on cell viability⁹. By exposing cells to a range of concentrations, the dose-response relationship can be assessed, which is vital for drug screening and toxicology studies. This relationship reveals the potency and toxicity of substances.

A common method for generating these curves is serial dilution, which prepares a series of concentrations that span multiple orders of magnitude. This procedure helps establish the connection between the concentration of a substance and the resulting cellular response.

The IC₅₀ is the concentration of a treatment required to reduce cell viability by 50%. This value can be determined either visually from the dose-response curve or by using specialized software to fit the data to a sigmoidal curve. A lower IC₅₀ indicates a more potent drug or higher toxicity for toxic substances.

Figure 1. MTT assay of Bortezomib in multiple myeloma cells.

An MTT assay kit ab211091 was used to assess the cell viability of myeloma cells resistant to bortezomib (a proteasome inhibitor used as a cancer therapeutic).

Cells previously treated with 10 nM bortezomib for 1.5 months (RPMI8226-bortezomib) had significantly improved viability, compared to the parental untreated cell line, when treated for 72 h with varying concentrations of bortezomib.

Interpreting absorbance readings

Absorbance readings provide direct insights into cell viability and metabolic activity.

After subtracting background absorbance from blank wells, a higher absorbance indicates a greater number of viable, metabolically active cells. This correlates with higher cell viability or growth, as purple formazan is proportionally produced by living cells undergoing metabolic activity.

In contrast, lower absorbance suggests a reduction in viable cells or impaired metabolic activity. This could indicate cytotoxicity (cell death) or inhibited cell growth due to the treatment.

While the MTT assay primarily measures cellular metabolic activity as an indicator of cell proliferation and viability, it is important to recognize that metabolic activity can also be influenced by factors other than cell viability.

For example, stress responses or drugs that alter metabolic pathways can interfere with MTT reduction, even in viable cells. Therefore, to achieve a deeper understanding of cell health, it is recommended to complement the MTT assay with other tests, such as cytotoxicity or apoptosis assays, which assess different aspects of cell function and integrity, providing a more holistic view of cellular responses to treatments.

Common applications of MTT assays

The MTT assay is a versatile technique with a wide range of applications, including drug discovery, cytotoxicity testing, and cell proliferation measurement^10,11,12.

Drug screening

The MTT assay plays an important role in drug discovery by identifying compounds with specific biological activities, such as inhibiting cancer cell growth, modulating immune system responses, and targeting specific cellular pathways involved in disease¹².

The MTT assay can be integrated into automated platforms, enabling the rapid screening of large compound libraries. The assay’s colorimetric nature allows for the simultaneous processing of multiple samples. By exposing cells to varying drug concentrations, dose-response curves are generated, and IC₅₀ values can be calculated. These values are essential for determining the optimal therapeutic concentration range for new drugs.

Cytotoxicity studies

MTT assays are widely used to assess the cytotoxicity of various agents, including environmental pollutants, nanoparticles, radiation exposure, and chemotherapeutic drugs. The assay provides valuable insights into the impact of pollutants on cell health across different organisms, aiding in environmental toxicology studies.

It also plays a vital role in radiation biology by evaluating the radiosensitivity of cancer cells, thus helping optimize radiation therapy protocols. As a cost-effective and simple method, the MTT assay offers an efficient way to evaluate cellular toxicity, providing essential data for risk assessments and guiding strategies to mitigate harmful exposures.

Cell proliferation analysis

MTT assays can be used to monitor cell proliferation in response to various stimuli, including growth factors, cytokines, and changes in culture conditions such as nutrient availability or temperature. The assay can be used to assess normal cell growth and the dysregulation of proliferation, particularly in cancer.

Additionally, MTT assays can be used in tissue engineering and regenerative medicine, where they are used to track the growth and viability of cells seeded on biomaterials and scaffolds. This is important for developing functional tissue replacements and ensuring the success of regenerative therapies.

Other applications

MTT assays are valuable for exploring cellular metabolism and physiology by examining how cells adapt to changes in their microenvironment, such as nutrient availability, oxygen levels (hypoxia), and temperature variations.

Additionally, MTT assays play a significant role in personalized medicine, particularly in cancer treatment. By assessing the sensitivity of primary cells, such as patient-derived tumor cells, to various therapeutic agents, the assay helps tailor treatment strategies. This enables clinicians to select the most effective therapies based on how an individual’s tumor cells respond to drugs, improving treatment outcomes.

Comparison with other cell viability assays

MTT assays are one among various cell viability assays, each with its pros and cons ^13,14. In the following table, the MTT assay is compared with other widely employed assays, such as CCK-8, WST-1, and LDH.

Troubleshooting and key considerations for MTT assay reliability

The accuracy of MTT assays is highly dependent on proper optimization and awareness of potential issues¹⁵.

Common issues in the MTT assay

Incomplete solubilization of formazan crystals

Incomplete solubilization of formazan crystals can occur due to insufficient solvent volume, inadequate mixing, or improper solvent composition, such as the absence of SDS or buffering agents.

Solvents like 5% SDS in buffered DMF or DMSO should be used to effectively dissolve the formazan and maintain its stability. Orbital shaking for 15-30 min or pipette mixing is recommended to ensure complete dislodging of the crystals. Additionally, full solubilization should be checked microscopically before absorbance measurements are taken.

Interference from test compounds

Interference from test compounds can occur when colored compounds or those with strong reducing or oxidizing properties interact with the MTT reagent or formazan product, leading to false positives or false negatives. To address this, control wells containing only the test compound (without cells) should be included to assess potential interference. If interference is significant, alternative assays that are less susceptible to this issue should be considered.

High background absorbance

High background absorbance may occur in wells without cells, potentially caused by contamination of the culture medium with reducing agents (such as phenol red) or serum components, microbial contamination, or degradation of the MTT solution. To mitigate this, fresh, high-quality reagents should be used, and a serum-free medium should be employed during incubation. Strict adherence to aseptic techniques is essential to prevent contamination. Additionally, absorbance from blank wells should be subtracted to correct for background noise.

Edge effects

Edge effects can occur when cells in the outer wells of a microplate experience different conditions, such as temperature fluctuations or evaporation, compared to those in the inner wells, leading to variability in results. To minimize this, only the inner wells should be used for experimental samples. The outer wells should be filled with sterile PBS or culture medium to maintain uniform conditions. Consistent handling and incubation conditions should also be ensured to prevent discrepancies between wells.

Replicate variability

Variability between replicates can arise due to inconsistent cell seeding, pipetting errors, uneven incubation times, or metabolic variability. To reduce this, cell seeding density should be optimized to ensure consistent cell growth across wells. Pipettes should be calibrated, and careful mixing of cell suspensions should be performed to ensure even distribution. Increasing the number of replicate wells can also improve statistical power, enhancing the reliability of the results.

MTT toxicity

MTT toxicity to cells can occur when excessively high concentrations of MTT inhibit cellular metabolism or cause cell death. To mitigate this, the MTT concentration should be optimized for the specific cell line used, with a starting concentration of 0.5 mg/mL recommended. Adjustments can be made based on the expected metabolic activity of the cells. Additionally, incubation times should be carefully controlled to avoid toxicity, ensuring that cells remain viable throughout the assay.

Tips for reliable MTT assay results

• Cells should be seeded during the exponential growth phase at densities that ensure healthy, metabolically active cells throughout the assay.
• The MTT stock solution should be freshly prepared by dissolving MTT powder in sterile PBS and filtering it through a 0.2 µm filter to remove contaminants.
• For storage, aliquots should be kept at -20°C, avoiding repeated freeze-thaw cycles.
• Using a serum-free medium minimizes interference from serum components, and if serum is needed, consistent concentrations should be maintained across all samples.
• Incubation times should be consistent for all samples to ensure uniform formazan production.
• Accurate pipetting is essential to avoid errors and ensure correct volumes of cells and reagents are dispensed.
• Solubilization of formazan crystals requires an appropriate solvent volume and thorough mixing, ideally with an orbital shaker in the dark for the recommended duration.
• Absorbance should be measured accurately at the recommended wavelength, typically 570 nm (or 590 nm). Readings should be taken within 1 h after solvent addition to avoid signal decay.
• Appropriate controls, including blank wells, untreated control wells, positive control wells, and vehicle control wells, should be included for reliable data interpretation.
• Biological and technical replicates, with at least 3-5 wells per condition, are recommended for statistical reliability.
• If phenol red is present, background absorbance should be corrected using a reference wavelength or by using a phenol red-free medium.
• Interactions with test compounds should be carefully evaluated by performing additional control experiments, and alternative assays should be considered if significant interference is detected.

FAQs

What are the limitations of the MTT assay?

The MTT assay measures metabolic activity, and not cell viability directly, and can be affected by factors such as mitochondrial dysfunction or interference from test compounds. It may also show variability between cell types and culture conditions.

What are some alternatives to the MTT assay for measuring cell viability or proliferation?

Alternatives to the MTT assay for measuring cell viability or proliferation include CCK-8, WST-1, resazurin-based assays like Alamar blue, and ATP-based luminescence assays. These alternatives may provide higher sensitivity, greater accuracy, or reduced interference from test compounds, depending on the specific experimental conditions and requirements.

References

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11. Surin, A. M., Sharipov, R. R., Krasil’Nikova, et al.  Disruption of functional activity of mitochondria during MTT assay of viability of cultured neurons. Biochemistry (Moscow). 82, 737-749 (2017)
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14. Skrzydlewski, P., Twarużek, M., and Grajewski, J. Cytotoxicity of mycotoxins and their combinations on different cell lines: a review. Toxins. 14(4), 244 (2022).
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