MTT assay

MTT assay is a coloritmetric assay to measure cell survival. It is based upon the ability of viable cells to convert MTT (yellow) to a formazan product (purple) which can be quantified using a spectrophotometer. The reaction is driven by the enzyme NAD(P)H-dependent cellular oxidoreductase that resides in mitochondria (Figure 1).

Figure 1 The yellow MTT is converted by the mitochondrial reductase into the purple formazan.

The protocol of an MTT assay works as follows:

  • Plate cells in 96 well plates at a density of 1,000 to 100,000 per well
  • Incubate for 24 hours to enable cells to adapt to the new conditions
  • Add MTT reagent
  • Incubate for 2 hours at 37 °C until purple precipitates are visible in the wells
  • Add detergent to dissolve the formazan crystals
  • Record absorbance at 570 nm

It's important to always include a negative control which contains either plain cell culture medium or the solvent of a potential drug tested within an MTT assay. MTT is then added to all conditions (wells) and the negative control later used as the reference value when analyzing the results.