Neutral red uptake assay for the estimation of cell viability/cytotoxicity

Nat Protoc. 2008;3(7):1125-31. doi: 10.1038/nprot.2008.75.


The neutral red uptake assay provides a quantitative estimation of the number of viable cells in a culture. It is one of the most used cytotoxicity tests with many biomedical and environmental applications. It is based on the ability of viable cells to incorporate and bind the supravital dye neutral red in the lysosomes. Most primary cells and cell lines from diverse origin may be successfully used. Cells are seeded in 96-well tissue culture plates and are treated for the appropriate period. The plates are then incubated for 2 h with a medium containing neutral red. The cells are subsequently washed, the dye is extracted in each well and the absorbance is read using a spectrophotometer. The procedure is cheaper and more sensitive than other cytotoxicity tests (tetrazolium salts, enzyme leakage or protein content). Once the cells have been treated, the assay can be completed in <3 h.

MeSH terms

  • Cell Count / methods*
  • Cell Survival / physiology*
  • Colorimetry / methods
  • Lysosomes / metabolism
  • Neutral Red / metabolism
  • Neutral Red / pharmacokinetics*
  • Spectrophotometry / methods*
  • Toxicity Tests / methods


  • Neutral Red