title: Use of the viability reagent PrestoBlue in comparison with alamarBlue and MTT to assess the viability of human corneal epithelial cells.
abstract: BACKGROUND
  PrestoBlue is a new resazurin based reagent to assess cell viability and cytotoxicity. It is claimed to be a fast and highly sensitive assay. Here, we compared PrestoBlue, alamarBlue, and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazonium bromide (MTT) in assessing cell viability of human corneal epithelial cells (HCEC), and investigated the effect of plate color, reading mode, and plate storage on the performance of PrestoBlue assay.
  
  
  METHODS
  The viability of different numbers of healthy HCEC and the toxicity of various chemicals on HCEC were evaluated using PrestoBlue (fluorescence), alamarBlue (fluorescence), and MTT (absorbance). The sensitivities of the three assays were compared. In the PrestoBlue assay, three plate colors and two reading modes were used and compared in assessing the toxic effect of sodium dodecyl sulfate (SDS). The PrestoBlue solutions after reaction were stored and measured on day 1, 2, 3, 5, and 7. The fluorescence readings obtained on different days were then compared.
  
  
  RESULTS
  Both PrestoBlue and alamarBlue were able to detect 5000 healthy cells after 30min incubation and 1000 cells after 1h, 2h, and 4h incubation; while MTT was able to detect 5000 cells after 3h incubation. In the assessment of the toxicity of various chemicals, PrestoBlue and alamarBlue performed similarly. There was no significant difference between the results obtained by these two reagents. All the three plate colors and two reading modes showed similar results in the PrestoBlue assay in assessing the toxicity of SDS. Plate storage up to 7days did not affect the result of the PrestoBlue assay.
  
  
  CONCLUSIONS
  Our study suggests that in evaluating the viability of HCEC, PrestoBlue is more sensitive than MTT, but similar to alamarBlue. The plate color, reading mode and plate storage up to 7days did not affect the performance of the PrestoBlue assay.
authors.affiliation: Optometry and Vision Science, University of Waterloo, Waterloo, ON N2L 3G1, Canada. Electronic address: melinda.ml.xu@gmail.com.
authors.forename: Manlong
authors.initials: M
authors.lastname: Xu
authors.affiliation: Centre for Contact Lens Research, Optometry and Vision Science, University of Waterloo, Waterloo, ON N2L 3G1, Canada.
authors.forename: David J
authors.initials: DJ
authors.lastname: McCanna
authors.affiliation: Optometry and Vision Science, University of Waterloo, Waterloo, ON N2L 3G1, Canada.
authors.forename: Jacob G
authors.initials: JG
authors.lastname: Sivak
mesh_terms: D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004728:Endothelium, Corneal / Q000166:cytology / Q000187:drug effects*; D005453:Fluorescence; D006801:Humans; D007202:Indicators and Reagents / Q000737:chemistry / Q000494:pharmacology*; D010078:Oxazines / Q000737:chemistry / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D013778:Tetrazolium Salts / Q000737:chemistry / Q000494:pharmacology*; D013844:Thiazoles / Q000737:chemistry / Q000494:pharmacology*; D014966:Xanthenes / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D007202:Indicators and Reagents; D010078:Oxazines; D013778:Tetrazolium Salts; D013844:Thiazoles; D014966:Xanthenes; C005843:resazurin; C022616:thiazolyl blue
keywords: AlamarBlue; Cell viability; Human corneal epithelial cells; MTT; PrestoBlue; Toxicity