MTT assay protocol

Introduction

The MTT assay is a cornerstone method in cell biology for evaluating cell viability and proliferation. It is especially valuable in drug discovery, toxicology, and cancer research. This protocol simplifies the process, guiding researchers through reagent preparation, incubation, and absorbance measurement. The assay’s principle is based on the enzymatic reduction of the MTT substrate by mitochondrial dehydrogenases in living cells, producing a quantifiable colorimetric signal. Only living cells are able to convert MTT into a colored formazan product, distinguishing them from dead or non-viable cells. With its straightforward workflow and compatibility with high-throughput formats, the MTT assay remains a preferred choice for researchers seeking accurate and reproducible data on cellular responses to treatments and environmental stressors.

Background and principles

The MTT assay utilizes the yellow tetrazolium salt MTT, which is reduced by mitochondrial enzymes in live cells to form insoluble purple formazan crystals. This reduction is primarily driven by succinate dehydrogenase and depends on enzyme activity within the cells, reflecting mitochondrial integrity and overall metabolic activity. The intensity of the purple color correlates with the number of viable cells and is measured spectrophotometrically, typically at 570–590 nm. A reference wavelength, such as 630 nm, can be used to correct for background absorbance and improve accuracy. Accurately measuring absorbance values is crucial, as these values directly reflect cell viability in the assay. The assay is sensitive to cytotoxic agents and provides quantitative data on cell health. It is essential to use serum-free media during incubation to avoid interference, as culture medium components can affect the solubilization and detection of the formazan product, potentially impacting the accuracy of absorbance readings. Proper solubilization of formazan is critical for accurate absorbance readings.

Stage 1 - Reagent preparation

Steps

MTT is soluble in water (10 mg/mL), ethanol (20 mg/mL), and buffered salt solutions and culture media (5 mg/mL). We recommend using a 5 mg/mL solution in PBS.

Prepare MTT solution.

• Mix by vortexing or sonication.
• Filter sterilize solution after adding MTT.
• Store MTT solution at -20°C (stable for at least 6 months).

Prepare MTT solvent 4 mM HCl, 0.1% NP40 in isopropanol.

Stage 2 - Assay protocol

• Serum or phenol red present in the culture medium can generate background. If your sample contains serum or phenol red, set up sample background controls: 50 µL MTT reagent + 50 µL cell culture media (no cells).
• Prepare parallel well(s) as solvent control and use same volume of solvent as for the treated cells.

Steps

Discard media from cell cultures.

• For adherent cells, carefully aspirate the media.
• For suspension cells, spin the 96 well plate at 1,000 x g, 4°C for 5 minutes in a microplate-compatible centrifuge and carefully aspirate the media.

Add 50 µL of serum-free media and 50 µL of MTT solution into each well.

Incubate the plate at 37°C for 3 hours.

After incubation, add 150 µL of MTT solvent into each well.

Wrap plate in foil and shake on an orbital shaker for 15 minutes.

Read absorbance at OD=590 nm.

Stage 3 - Data analysis

Steps

Average the duplicate reading for each sample.

Subtract the culture medium background from your assay reading. This is the corrected absorbance.

For cell counting, a standard curve can be established with known cell number and fixed incubation times with the assay reagent.

Average the duplicate reading for each sample.

Subtract the culture medium background from your assay readings. This is the corrected absorbance.

Calculate percentage cytotoxicity with the following equation, using corrected absorbance.

% cytoxicity = (100 x (control - sample))

Membrane integrity, metabolic activity, and enzymatic activity

Assessing cell viability requires a comprehensive understanding of several key cellular parameters: membrane integrity, metabolic activity, and enzymatic activity. Membrane integrity is a fundamental indicator of cell health, as intact membranes are essential for maintaining cellular homeostasis. Viability assays often use nucleic acid binding dyes, such as propidium iodide (PI) and 7-aminoactinomycin D (7-AAD), to identify cells with compromised membranes—these dyes penetrate only damaged cells, providing a clear distinction between live and dead cell populations.

Metabolic activity is another critical marker, reflecting the ability of cells to carry out essential biochemical processes. Cell proliferation assays like MTT, XTT, and WST-1, as well as resazurin-based assays, measure the reduction of tetrazolium salts or resazurin by metabolically active cells. The signal generated in these assays is directly proportional to the number of viable cells, making them invaluable for determining cell viability and monitoring cell growth.

Enzymatic activity, particularly that of enzymes such as lactate dehydrogenase (LDH) or cellular esterases, provides additional insight into cell health. LDH release assays, for example, detect cell death by measuring the enzyme’s presence in the culture medium, indicating loss of membrane integrity. By combining these approaches, researchers can accurately assess the effects of cytotoxicity assays on cell populations, distinguish between healthy and damaged cells, and gain a deeper understanding of cellular responses in various assay conditions.

Comparison to other methods

Compared to other viability assays like XTT, MTS, and resazurin-based methods, the MTT assay offers robust sensitivity and reproducibility. While MTS and XTT produce soluble formazan products, MTT requires a solubilization step, which can be a limitation but also allows for more controlled quantification. Resazurin assays are faster and less toxic but may be less specific to mitochondrial activity. Most assays can be multiplexed with different detection methods, such as fluorescent or luminescent assays, to provide comprehensive data in complex experimental workflows. The MTT assay is an endpoint assay and does not allow for continuous monitoring of cell viability, unlike some real-time fluorescent assays that enable dynamic assessment over time. The MTT assay’s reliance on mitochondrial enzymes makes it particularly suitable for detecting subtle changes in metabolic activity, but its performance can vary depending on the cell types used, and optimization may be required for different cell types.

Applications of cell viability assays

The MTT assay is widely applied in cell biology, pharmacology, and toxicology to assess cell viability, proliferation, and cytotoxicity. Cell viability and activity can be assayed using the MTT protocol, providing reliable measurements of cellular responses. It is instrumental in drug screening, evaluating the effects of compounds on cell health, and studying apoptosis and necrosis. Researchers use it to monitor cellular responses to environmental stressors, genetic modifications, and therapeutic interventions. This protocol provides instructions for both adherent and suspension cells, making it versatile across various experimental setups. Its quantitative nature allows for the generation of dose-response curves, where compounds are typically tested at various concentrations to assess their effects on cell viability, and for standard cell growth profiles, facilitating high-throughput screening and comparative analysis.

Limitations of cytotoxicity assays

Despite its widespread use, the MTT assays have their limitations. The requirement for solubilizing formazan crystals adds complexity and potential variability. Interference from serum, phenol red, and reducing agents like ascorbic acid can skew results. The assay is also sensitive to cell density and incubation time, which must be optimized for each cell type. Additionally, adjusting the pH of the solubilization solution can enhance the maximum absorbance of the formazan product, improving detection sensitivity. MTT reduction occurs in multiple cellular compartments, not exclusively mitochondria, which may affect specificity. Abcam’s protocol addresses many of these issues with detailed troubleshooting and optimized reagents, but careful experimental design remains essential for reliable outcomes.

Troubleshooting

Common issues in the MTT assay include low absorbance, high background, and inconsistent results. These can stem from using ice-cold reagents, incorrect wavelength settings, or interference from serum and phenol red. Ensure reagents are warmed to room temperature and use serum-free media during incubation. For adherent cells, gently remove MTT reagent before adding solvent; for suspension cells, add solvent directly to avoid cell loss. If formazan crystals do not dissolve fully, increase shaking time or pipette gently. Abcam’s protocol includes specific solutions for each problem, helping researchers maintain assay accuracy and reproducibility.