Comparison of alamar blue and MTT assays for high through-put screening

Toxicol In Vitro. 2004 Oct;18(5):703-10. doi: 10.1016/j.tiv.2004.03.012.


The performance of alamar blue and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) cell viability assays in a high through-put format were compared. A total of 117 drugs chosen for their wide range of therapeutic areas were screened at 10 microM using both assays in human hepatoma cell line HepG2. Except for terfenadine and astemizole, which performed consistently in both assays, the alamar blue assay was slightly more sensitive than the MTT assay for most compounds. The MTT assay was less sensitive detecting an effect for daunorubicin and trifluoperazine. Seven drugs, astemizole, daunorubicin, ellipticine, fluphenazine, terfenadine, thioridazine and trifluoperazine, had percent viability results of 55% or less in the alamar blue assay at the single point screen. These were re-tested in both assays for reconfirmation of cytotoxicity and determination of the EC50 values. Except for daunorubicin, the EC50 values were comparable in both assays. Based on these results and the Z'-factor assessment of assay quality, both assays provided useful information to identify in vitro cytotoxic drugs at early stages of drug candidate selection. However, careful interpretation of data is warranted due to the possibility of false positive or negative results caused by inducers and/or inhibitors of metabolic enzymes that are responsible for transformation of cell toxicity end points, as we demonstrated using dicumarol.

Publication types

  • Comparative Study

MeSH terms

  • Carcinoma, Hepatocellular / drug therapy
  • Carcinoma, Hepatocellular / metabolism*
  • Cell Line, Tumor
  • Cell Survival / drug effects
  • Dose-Response Relationship, Drug
  • Drug Evaluation, Preclinical / methods*
  • Formazans / metabolism*
  • Humans
  • Indicators and Reagents / metabolism*
  • Oxazines / metabolism*
  • Reproducibility of Results
  • Tetrazolium Salts / metabolism*
  • Xanthenes / metabolism*
  • Xenobiotics / toxicity*


  • Formazans
  • Indicators and Reagents
  • Oxazines
  • Tetrazolium Salts
  • Xanthenes
  • Xenobiotics
  • resazurin
  • MTT formazan