MTS Assay: Switching On the “Traffic Light” of Cellular Life

The Absin MTS Cell Proliferation Assay is based on a water-soluble tetrazolium salt that is bioreduced by dehydrogenases in viable cells to generate a soluble formazan product. The absorbance at 490–500 nm is directly proportional to the number of living cells, providing a quantitative read-out of cell number and metabolic activity.

This technology has become a gold-standard tool in biomedical research for evaluating proliferation, cytotoxicity and cellular viability.

01 Detection Principle & Mechanism

MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) is a next-generation water-soluble tetrazolium salt.

It is reduced by succinate dehydrogenase and other NAD(P)H-dependent oxidoreductases in active mitochondria to a soluble, orange-colored formazan.

The reaction is simple: metabolically robust cells generate more formazan, deepening the color; stressed, apoptotic or necrotic cells produce less, yielding a lighter hue.

This colorimetric correlation with cellular redox capacity makes MTS an intuitive and reliable indicator of cell viability.

02 Key Advantages: Why Choose MTS?

Compared with conventional MTT, MTS offers significant technical improvements:

The water-soluble feature makes MTS ideal for high-throughput screening and continuous monitoring on the same plate without solvent-induced artefacts or cell damage.

03 Which Assays Can Benefit from MTS?

MTS is broadly applied across biomedical research:

• Drug discovery: dose–response curves, IC₅₀ determination for anti-cancer, anti-microbial or anti-viral compounds.
• Toxicology: evaluation of environmental pollutants, heavy metals, nanomaterials.
• Cell biology: quantification of growth-factor or cytokine activity, gene-knockout effects on proliferation.
• Tissue engineering & organoids: metabolic profiling of 3-D constructs; a 2024 study compared MTS, WST-8 and ATP assays in human chondrocyte micro-tissues.

04 Application in 2-D vs 3-D Cultures

2-D cultures: seed 5–10 × 10³ cells per 100 µL in 96-well plates. After treatment add 10 µL MTS reagent, incubate 1–4 h, and read A_{490–500 nm}. A standard curve converts absorbance to cell number or relative viability is calculated versus untreated controls.

3-D cultures: penetration of MTS into large spheroids is limited; a 2024 study showed that ATP assays remained linear across all micro-tissue sizes whereas MTS linearity was restricted. Prolonged (>6 h) MTS incubation can also become cytotoxic—an important consideration when designing 3-D experiments.

05 Assay Optimisation & Quality Control

Optimal seeding density (usually 5–10 × 10³ cells/well) and edge-effect controls (fill peripheral wells with sterile PBS) should be pre-determined. Include blank (medium + MTS only), negative (vehicle) and positive (known inhibitor) controls. Reducing agents in some drugs can interfere; always run drug-only blanks for background subtraction.

06 Limitations & Future Directions

MTS reports global metabolic activity, not absolute cell number, and can be influenced by mitochondrial dysfunction or metabolic reprogramming. Penetration depth in large 3-D constructs is limited, potentially underestimating inner-core viability. Real-time kinetic imaging is not possible once the colored product accumulates. Future strategies may couple MTS with ATP-based or live-cell fluorescence imaging to provide complementary, orthogonal read-outs.

Despite these caveats, MTS remains a versatile, user-friendly “traffic-light” for cellular fitness in both routine 2-D screens and emerging 3-D models.

Recommended Absin MTS Cell Proliferation Assay Kit:

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