title: Alamar blue reagent interacts with cell-culture media giving different fluorescence over time: potential for false positives.
abstract: BACKGROUND
  The cell viability assay by alamar blue is based on the principle of reduction of the non-fluorescent reagent (resazurin) to a fluorescent compound (resarufin) by the intracellular reducing environment of living cells over time. In the present study, we have for the first time shown that even in the absence of cells, there occurs significant interaction between alamar blue and cell-culture media causing an increase in fluorescence.
  
  
  METHODS
  We have used Opti-MEM, DMEM and 1:1 DMEM:Opti-MEM as three different media and determined the changes in their relative fluorescence units (RFUs) over time after the addition of 10% (v/v) alamar blue using two-way repeated measures analysis of variance (RM-ANOVA) followed by Tukey's post-hoc test.
  
  
  RESULTS
  Our results show that upon the addition of alamar blue, there occurs a significant increase in RFUs in all the three media over time along with a significantly higher RFU for the Opti-MEM overall (p<0.05). We also show that the time-dependent change in RFU of 1:1 DMEM:Opti-MEM was more gradual compared to that of the other two media.
  
  
  CONCLUSIONS
  These findings indicate that the reagent can itself interact with the media causing significantly different fluorescence over time in a manner independent from the effect of intracellular reducing environment of living cells on alamar blue. In addition our results indicate that fluorescence varies as a function of incubation time with the reagent. These findings signify the need for routine subtraction of the background fluorescence of media-only with alamar blue reagent during measurement of cell viability by this method in order to determine an accurate measurement of cell viability.
authors.affiliation: Department of Neuroscience, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064, USA. Electronic address: soumyabrata.munshi@my.rfums.org.
authors.forename: Soumyabrata
authors.initials: S
authors.lastname: Munshi
authors.affiliation: Department of Cellular and Molecular Pharmacology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064, USA. Electronic address: robert.twining@rosalindfranklin.edu.
authors.forename: Robert C
authors.initials: RC
authors.lastname: Twining
authors.affiliation: Department of Neuroscience, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064, USA; Department of Pharmaceutical Sciences, College of Pharmacy, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064, USA. Electronic address: russell.dahl@rosalindfranklin.edu.
authors.forename: Russell
authors.initials: R
authors.lastname: Dahl
mesh_terms: D016477:Artifacts; D003470:Culture Media / Q000737:chemistry*; D005189:False Positive Reactions; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D010078:Oxazines / Q000737:chemistry*; D013997:Time Factors; D014966:Xanthenes / Q000737:chemistry*
chemical_list: D003470:Culture Media; D005456:Fluorescent Dyes; D010078:Oxazines; D014966:Xanthenes; C005843:resazurin
keywords: Alamar blue assay; Media; Reduction; Relative fluorescence unit; Resazurin; Time