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 1The 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
• 培养24小时,使细胞适应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.