In vitro cytotoxicity – MTT & NR

Aim: To assess the potential for basal cell cytotoxicity in vitro.

Background information: Cell viability tests allow assessing the interference of test item with basic cellular functions, that can ultimately lead to cell cytotoxicity and tissue damage. Prospective cytotoxicity screening tests such as the colorimetric MTT and Neutral Red (NR) assays are used to approximate the safety characteristics of individual compounds or lead pharmaceutical series early in the drug discovery process, allowing to select those with better safety characteristics.

The MTT assay is based on the ability of living functional cells to metabolically cleave the yellow coloured and water-soluble tetrazolium ring (MTT), into purple coloured non-water-soluble formazan crystals, this being an indicator of the integrity and functional level of the mitochondria.

The Neutral Red (NR) assay is based on the inclusion of this non-toxic colorant into the lysosomes of viable cells. Changes in the entry and/or retention of this colorant within the lysosomes are indicative of cell and/or lysosomal membrane damage or interference with the normal energy requiring process of endocytosis.

The level of light absorbance by the purple formazan and NR can be related to the viability and number of cells present.

Cell type

  • HepG2 cells
  • Other cell types on demand


Experimental procedure

  • Top concentration: 100 µM (or on demand)
  • 20 h treatment; 37ºC, 5% CO2
  • Microscopic morphological changes observation
  • Treatment medium discarded
  • 3 h exposure in MTT or NR medium; 37ºC, 5% CO2
  • Absorbance at 550 nm

Delivered results:

  • Test item concentration producing a 50% decrease
    in cell viability