MTT assay

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The MTT assay is a simple test to estimate how many cells are alive based on their metabolism. In living cells, certain enzymes use NAD(P)H to convert the yellow dye MTT into a purple, insoluble substance called formazan. The more living cells you have, the more purple color is formed.

How the test works
- Add MTT to the cells and incubate (usually kept in the dark because MTT is light-sensitive).
- Viable cells reduce MTT to purple formazan.
- Since formazan is insoluble, you dissolve it with a solvent (such as DMSO, an acidified ethanol solution, or a detergent solution) to make a colored liquid.
- Measure the color with a spectrophotometer at a wavelength typically between 500 and 600 nm. Stronger color means more viable cells.

What about related dyes
- XTT, MTS, and WST dyes are similar tests with some differences:
- XTT: forms a water-soluble formazan, often more sensitive.
- MTS: a one-step test you can add directly to cells, but may be more prone to interference from colored substances.
- WSTs (including WST-1 and WST-8): generally reduced outside the cells and also give water-soluble formazan, usually easier to read and gentler on cells.
- WSTs let you read without a separate solubilization step and can be less toxic to cells, but results should still be interpreted with proper controls.

What the reading tells you
- The assay reflects cellular metabolic activity, mainly NAD(P)H-dependent enzymes in the cell.
- It’s often used to assess cytotoxicity (cell death) or cytostasis (slowed or stopped cell growth) caused by drugs or toxins.
- In some situations, changes in metabolism (not just cell number) can affect the result, so controls are important.

Important tips
- Keep the assay conditions consistent, as metabolism can change results even if cell viability doesn’t.
- For cells grown on 3D materials or thick scaffolds, diffusion and light penetration can affect readings, so interpretation may require extra care.
- Always compare treated samples to appropriate controls and consider confirming results with additional observations (like microscope checks).

How to quantify viability
- A common formula is: viability (%) = (absorbance of sample / absorbance of control) × 100.

In short, the MTT and related tetrazolium dye tests are practical, easy ways to gauge how many cells are metabolically active, with several dye options offering different conveniences and readouts.