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Cell Viability Assays: MTT, Resazurin and ATP, and How Compounds Interfere
Most cell-based peptide studies include a viability or proliferation readout. The number looks like a head count of living cells, but it is not. Each common assay measures a chemical activity that usually tracks cell number, and sometimes it does not.
Key takeaways
- MTT and similar assays measure reducing activity of cells.
- Resazurin is a fluorescent, reversible-sensor version.
- ATP assays measure energy status through luminescence.
- Some test compounds reduce the dyes directly and give false viability.
The tetrazolium family: MTT, MTS, XTT
MTT is a yellow tetrazolium salt that cells reduce to purple formazan crystals using NAD(P)H-dependent enzymes. The crystals must be dissolved before reading absorbance near 570 nm. MTS and XTT give soluble products, so they skip the dissolving step. All of them report metabolic reducing capacity, which tracks cell number only when metabolism per cell stays constant.
Resazurin (alamarBlue)
Resazurin is a blue, weakly fluorescent dye that cells reduce to pink, highly fluorescent resorufin. It is non-destructive in the short term, so cells can be measured repeatedly, and it is more sensitive than MTT. Again it measures reducing activity.
ATP assays
Luciferase-based reagents such as CellTiter-Glo lyse cells and produce light in proportion to ATP. ATP falls rapidly when cells die, so the readout is fast and sensitive, and less prone to chemical interference. It still measures energy state, which can change without cell death.
Membrane integrity assays
LDH release measures the enzyme lactate dehydrogenase leaking from cells with damaged membranes, and dyes such as propidium iodide enter only dead cells. They report death rather than growth.
How compounds create false signals
- Direct reducers. Thiol-containing molecules, ascorbic acid, and some polyphenols can reduce MTT or resazurin without any cells, raising the signal. Peptides with free cysteine residues are a possible source (see cysteine handling).
- Metals and complexes. Redox-active metal complexes may also react with dyes.
- Color and fluorescence. A colored or fluorescent test compound adds to the optical signal.
- Changes in metabolism. A compound that boosts mitochondrial activity can raise MTT signal without increasing cell number, and the reverse is also true.
- Serum and phenol red add background in some assays.
Controls that catch problems
- A cell-free well with medium, dye and the highest compound concentration.
- A parallel direct cell count or DNA-based assay (such as a nuclear stain) to confirm cell number.
- Vehicle controls matched in solvent and preservative, as in our controls guide.
- Two different assay chemistries for the key results.
Interpreting the curve
Viability curves are dose-response curves (see EC50 explained), so a stable top and bottom and enough points on each side are required. A flat line at 100 percent is not proof of safety if the assay cannot see the damage.
The bottom line
A viability readout is a chemical surrogate for a biological question. Match the assay to the question, check for interference with a cell-free control, and confirm key results with a second method.
Related reading
- Vehicle, Scrambled and Positive Controls in Peptide Studies
- Dose-Response Curves in Plain Language: EC50, Efficacy and Hill Slope
- Cysteine and Disulfide Bonds: A Handling Guide for Peptide Researchers
- Browse the full Research Library
For laboratory research use only. Not for human or veterinary use, and not intended to diagnose, treat, cure or prevent any condition. This article is educational and is not medical advice.