Cell viability assay · BEGINNER LESSON
MTT assay
The MTT assay is a color-based laboratory test that estimates cellular metabolic activity by measuring how living cells reduce the MTT dye into an insoluble purple product called formazan. The absorbance of the dissolved formazan is then used as a proxy for the number of metabolically active cells, but it does not measure viability perfectly.
What it is and what is measured
MTT stands for 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. In the assay, cells convert this tetrazolium salt into formazan, a purple, water-insoluble product. After the formazan is dissolved, its absorbance is measured with a reader, and that signal is used to estimate metabolic activity in the sample. Because metabolically active cells usually produce more signal, the assay is often used as an indirect readout for cell number, proliferation, or cell health.
[1] Limitations and Strengths of the SH‐SY5Y Neuroblastoma Cell Line ... [2] pmc.ncbi.nlm.nih.gov › articles › PMC12653452Advances in Cytotoxicity Testing: From In Vitro Assays to In ... [3] Revisiting ISO 10993‐5 In Vitro Cytotoxicity Standard Tests for ...
How it works
The main idea is enzymatic reduction: enzymes in cells, especially dehydrogenases linked to mitochondrial and cytosolic metabolism, reduce MTT into formazan. The signal therefore reflects combined cellular reduction capacity rather than a single organelle or a direct count of living cells. In practice, this makes the assay simple and widely used, but also vulnerable to outside influences such as medium composition, reagent concentration, and compounds that reduce the dye themselves.
[1] Limitations and Strengths of the SH‐SY5Y Neuroblastoma Cell Line ... [2] pmc.ncbi.nlm.nih.gov › articles › PMC12653452Advances in Cytotoxicity Testing: From In Vitro Assays to In ... [3] Revisiting ISO 10993‐5 In Vitro Cytotoxicity Standard Tests for ...
Why the result can be misleading
The MTT assay does not always separate true cell survival from other changes in metabolism. Some studies report reduction of MTT by extra-mitochondrial enzymes, and test compounds or medium components can interfere with the readout. The assay can also miss subtle or early toxicity compared with other methods, and it may not distinguish cytotoxic effects, which kill cells, from cytostatic effects, which slow cell growth without killing cells.
[1] Limitations and Strengths of the SH‐SY5Y Neuroblastoma Cell Line ... [2] pmc.ncbi.nlm.nih.gov › articles › PMC12653452Advances in Cytotoxicity Testing: From In Vitro Assays to In ... [3] Revisiting ISO 10993‐5 In Vitro Cytotoxicity Standard Tests for ...
Limits and best interpretation
Because of these limits, MTT should be treated as an assay of metabolic activity, not as a perfect direct measure of viability. Results are strongest when the MTT readout is supported by independent assays that examine other endpoints, such as membrane integrity, attachment, proliferation, or apoptosis. This is especially important when experimental conditions can change metabolism without causing the same change in cell number.
[2] pmc.ncbi.nlm.nih.gov › articles › PMC12653452Advances in Cytotoxicity Testing: From In Vitro Assays to In ... [3] Revisiting ISO 10993‐5 In Vitro Cytotoxicity Standard Tests for ... [1] Limitations and Strengths of the SH‐SY5Y Neuroblastoma Cell Line ...
SEE THE IDEA
How an MTT assay produces a signal
Yellow tetrazolium dye is placed on cells.
Metabolically active cells convert MTT to formazan.
The insoluble purple formazan is solubilized.
A reader measures color intensity as an indirect signal.
Each step leads to the next, but the final color signal reflects metabolic reduction capacity, not a perfect cell count.
The assay turns a chemical conversion by cells into a measurable color signal.
TRY IT YOURSELF
Test your understanding
Choose an answer, then check the explanation.
PUT IT TOGETHER
Match each concept to its meaning
Drag a label onto its description, or select a label and then select a description. This also works with a keyboard.
4 pairs to match
Sources and further reading
This general lesson was generated from public source material. It explains a concept; it does not interpret a private paper or provide medical advice.