MTT, Resazurin, or Trypan Blue: Which Cell Viability Assay Fits Your Work?

Cell viability assays are one of the most routine measurements in biological research, and yet the choice of assay format still catches people out. MTT, resazurin, and trypan blue exclusion each measure something slightly different, and the differences matter when you are trying to interpret a dose-response curve or compare results across experiments.

MTT: the established standard with a solubilisation step ¶

The MTT assay measures metabolic activity by detecting the reduction of a yellow tetrazolium salt to a purple formazan product by mitochondrial enzymes in living cells. It is well established, widely cited, and the protocol is familiar to most cell biology laboratories.

The limitation is the solubilisation step. The formazan product is insoluble and must be dissolved before the plate can be read, which adds a step and a potential source of variability. If the solubilisation is incomplete or inconsistent across wells, the absorbance readings will not accurately reflect cell number. Some compounds also interfere with the reduction reaction directly, which can give falsely high or falsely low viability readings independent of actual cell death.

Resazurin: simpler workflow, kinetic-compatible ¶

Resazurin-based assays work on a similar principle. A non-fluorescent dye is reduced to a fluorescent product by metabolically active cells. But the product is soluble, which removes the solubilisation step. The plate can be read directly after incubation, and because the reaction is continuous, the assay is compatible with kinetic reading over time.

Resazurin is generally more sensitive than MTT at low cell numbers and is less prone to compound interference, though it is not immune. It is a good default choice for cytotoxicity screening work where throughput matters and the compound library is chemically diverse.

Trypan blue: direct count, no metabolic assumption ¶

Trypan blue exclusion is the only method of the three that directly counts cells rather than inferring viability from a metabolic signal. Dead cells with compromised membranes take up the dye and appear blue under a microscope; live cells exclude it and appear clear. The count is direct and does not depend on metabolic activity.

The limitation is throughput. Manual counting with a haemocytometer is slow and operator-dependent. Automated cell counters improve consistency but add instrument cost. For experiments where you need a direct cell count alongside a viability percentage. For example, when seeding cells for a subsequent experiment. Trypan blue is the appropriate method. For high-throughput screening, it is not practical.

Choosing based on your experimental context ¶

If you are running a compound screen in 96- or 384-well format, resazurin is usually the most practical choice. If you need to compare your results to a large body of published literature that used MTT, MTT is worth the extra step. If you need an absolute cell count, use trypan blue. If you are unsure whether your compound interferes with the reduction reaction, run a parallel trypan blue count on a subset of wells to check.

The choice of viability assay is rarely critical if you are consistent within an experiment. Where it becomes important is when you are comparing results across experiments, across laboratories, or against published data. Knowing what each method actually measures helps you interpret those comparisons correctly.