The MTT assay is a colorimetric lab test that measures how many living cells are in a sample. It works by adding a yellow dye to cells and waiting for living cells to convert it into purple crystals. The deeper the purple color, the more living cells are present. It’s one of the most widely used tests in biomedical research for evaluating cell viability, drug toxicity, and cell proliferation.
How the MTT Assay Works
MTT stands for 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide. It’s a yellow, water-soluble compound called a tetrazolium salt. Because the molecule carries a positive charge and has a fat-soluble structure, it can slip through cell membranes and enter the interior of living cells.
Once inside, metabolically active cells break apart the core ring structure of the MTT molecule and convert it into formazan, a violet-blue crystal that doesn’t dissolve in water. The enzymes responsible for this conversion are oxidoreductases and dehydrogenases, which are active at multiple points in a cell’s energy-producing pathways, from sugar metabolism all the way to the mitochondrial electron transport chain. The key fuel for this reaction is NADH and NADPH, molecules that cells generate as part of normal metabolism. Dead or damaged cells produce little to no formazan because their metabolic machinery has shut down.
The core logic is simple: living cells produce purple crystals, dead cells don’t. More purple means more living cells.
Step-by-Step Protocol
The standard MTT protocol uses 96-well plates, small plastic trays with dozens of tiny wells that each hold a separate cell sample. This format lets researchers test many conditions at once. The typical steps are:
- Prepare the plate. Cells and any test compounds (such as a candidate drug) are placed in wells with a final volume of 100 µl per well.
- Expose cells. The plate is incubated for whatever treatment duration the experiment requires.
- Add MTT solution. Ten µl of MTT solution is added to each well, reaching a final concentration of 0.45 mg/ml.
- Incubate. The plate sits at 37°C for 1 to 4 hours while living cells convert MTT into purple formazan crystals.
- Dissolve the crystals. Because formazan is water-insoluble, a solubilization solution (100 µl per well) is added to dissolve the crystals into a uniform purple liquid.
- Read absorbance. A spectrophotometer measures how much light each well absorbs at 570 nm. Higher absorbance means more formazan, which means more living cells.
Dissolving the Formazan Crystals
The solubilization step is critical because undissolved crystals give inconsistent readings. Several solvents can do the job. DMSO (dimethyl sulfoxide) is one of the most common, often buffered with ammonia at pH 10. Other options include acidified isopropanol, ethanol, and dimethylformamide (DMF) with SDS, a detergent that helps break apart both the crystals and the cells. Research comparing different formulations found that DMF and DMSO buffered with ammonia and containing 5% SDS produced the most reliable results. Whichever solvent is used, thorough mixing is essential to ensure complete dissolution before the plate is read.
Calculating Cell Viability
Results are typically expressed as percent cell viability relative to untreated control cells. The formula is straightforward: divide the absorbance of the treated sample by the absorbance of the untreated control, then multiply by 100. A result of 50% means the treatment killed roughly half the cells. Researchers often subtract the absorbance of a blank well (containing medium and solvent but no cells) from both values first to remove background noise. This calculation is the basis for determining metrics like the IC50, the concentration of a drug that kills 50% of cells.
Common Applications
The MTT assay is a workhorse in drug discovery. When researchers screen hundreds of chemical compounds for potential anticancer activity, they typically grow cancer cells in 96-well plates, expose them to different drug concentrations, and run the MTT assay to see which compounds kill the most cells. The same approach is used in toxicology to test whether industrial chemicals, cosmetic ingredients, or environmental pollutants are harmful to cells.
Beyond toxicity testing, the assay is used to track cell proliferation. If you want to know whether a growth factor or nutrient stimulates cells to multiply, you can plate identical cell samples, treat some with the compound, and compare absorbance values after a few days. Higher readings in treated wells indicate faster growth.
Known Limitations and Pitfalls
The MTT assay measures metabolic activity, not cell number directly. That distinction matters because certain conditions can inflate or deflate metabolic activity without changing how many cells are actually alive.
One well-documented problem involves mitochondrial uncoupling agents. These are compounds that speed up the mitochondrial electron transport chain without producing useful energy. The faster chain activity converts more MTT into formazan, making it look like more cells are alive when they aren’t. Rottlerin, a compound studied for its effects on cancer cells, caused significant overestimation of cell viability in MCF-7 breast cancer cells through exactly this mechanism. The synthetic uncoupler FCCP produced the same artifact at comparable doses and exposure times. Because many natural and synthetic molecules, including certain polyphenols and steroid hormones, have uncoupling properties, this is not an edge case.
A separate issue involves redox-active compounds. Some plant extracts and polyphenols can directly reduce the MTT salt to formazan even in the absence of living cells, generating a purple signal with no biological meaning. Any experiment testing antioxidant-rich natural products should account for this possibility by running cell-free control wells.
Colored test compounds can also interfere. If a drug or extract absorbs light near 570 nm, it will add to the formazan signal and skew results upward.
Alternatives to the MTT Assay
The MTT assay’s biggest practical drawback is the solubilization step. Because formazan crystals don’t dissolve in water, you have to add a solvent, wait, and mix before reading. Newer tetrazolium-based assays eliminate this step entirely.
The CCK-8 assay uses a different tetrazolium salt called WST-8, which cells convert into a water-soluble orange formazan. No crystals form, so no dissolution step is needed. You simply add the reagent, incubate, and read. CCK-8 also offers higher detection sensitivity than MTT. However, it remains an endpoint method, meaning each measurement captures a single snapshot in time. It’s also susceptible to the same spectroscopic interference from colored drugs.
For experiments that need continuous monitoring, real-time cell analysis (RTCA) systems track cells growing on gold microchip electrodes. As cells proliferate or die, the electrical impedance across the electrode changes, providing a dynamic, label-free readout of cell status without adding any dye at all. The tradeoff is cost: RTCA equipment is substantially more expensive than a standard plate reader.
Despite these newer options, the MTT assay remains popular because it’s inexpensive, well-validated across decades of published research, and compatible with equipment found in virtually every cell biology lab.