Many strange MTT results come from the compound reducing the dye, or from formazan crystals lost during aspiration. One cell-free blank and a seeding curve catch most of them.
Range: Biochemical and cell-based assays3 minute readUpdated
| Problem | Likely cause | Fix |
|---|---|---|
| High signal in cell-free wells | Compound or medium reduces MTT | Blank each compound; consider another assay |
| Purple before adding cells | Contaminated or degraded MTT | Make fresh, filter and keep in the dark |
| Wells differ by position | Edge effects or uneven seeding | Fill edge wells with medium; mix cells as you seed |
| Crystals remain after solvent | Incomplete solubilisation | Shake longer; check solvent volume |
| Viability above 100% of control | Raised metabolism or dye reduction | Confirm by counting or an orthogonal assay |
| Flat curve at high cell number | Signal saturated | Seed fewer cells |
MTT measures reduction, and it cannot tell a cell’s enzymes from a reducing chemical. Antioxidants, thiols and many plant extracts reduce MTT directly, which makes treated cells look healthier than they are. Some anti-cancer drugs interfere as well.
MTT signal tracks cell number only over a limited range, which differs between cell lines. Seed a dilution series once for each line and pick a density that stays linear through the end of the treatment. Formazan also accumulates in lipid droplets, so lipid content can influence the signal between cell types. Compare treatments within one cell line rather than across lines.
If interference or handling keeps causing trouble, a water-soluble tetrazolium avoids the aspiration step and an ATP assay avoids dye reduction altogether. The MTT vs CCK-8 guide compares them, and the cell-based assay range covers the kits. For function rather than viability, see enzyme activity assay kits.
The usual causes are a compound or medium component reducing MTT directly, degraded MTT or microbial contamination. Cell-free wells containing the compound show whether the chemistry is the cause.
MTT measures metabolic activity, not cell number. A treatment can raise metabolism, or the compound may reduce MTT directly. Confirm with a cell count or a different assay.
Usually too little solvent or too little mixing. Shake the plate for longer and make sure the solvent volume covers the well contents.
Yes. Antioxidants, thiols and many plant extracts can reduce MTT without any cells, which falsely raises the signal.
For research use only. Protocols here are general outlines: follow your kit manual and your institution’s safety rules. See our editorial policy.
Products for this workflow
Enzyme activity and metabolite assay kits, and cell function products for viability, proliferation, apoptosis and cell cycle experiments.
Biochemical and cell assays
Viability assays measure metabolism, membranes or ATP, never cell number directly. Knowing which one you are measuring explains most odd results and tells you which assay to pick.
5 minute read
Biochemical and cell assays
Most enzyme activity kits turn a reaction you cannot see into a colour change you can measure. Once you know the two or three ways they do it, choosing a kit and reading the result gets much easier.
4 minute read
Biochemical and cell assays
Succinate dehydrogenase is the only enzyme in both the Krebs cycle and the electron transport chain, which makes its activity a direct read on mitochondrial function. Here is how the assay works and how to get numbers you can trust.
5 minute read
Send the product names or catalogue numbers, quantities and delivery country. We'll confirm availability, lead time and the documents for biochemical and cell-based assays.