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Manlong Xu, David J McCanna, Jacob G Sivak; Use of the Viability Reagent PrestoBlue in Comparison with AlamarBlue and MTT to Assess the Health of Human Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):495.
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© ARVO (1962-2015); The Authors (2016-present)
To compare PrestoBlue with alamarBlue and MTT in assessing the cell viability of human corneal epithelial cells (HCEC), and to evaluate the toxic effect of ultraviolet (UV) radiation on cultured HCEC using PrestoBlue assay.
The metabolic activity of HCEC was measured using PrestoBlue, alamarBlue, and MTT. Three sets of experiments were done in this study. In set 1, different numbers of HCEC, ranging from 20 to 50000, was tested using PrestoBlue and alamarBlue with 4 different incubation times, and using MTT with one standard incubation time. In set 2, HCEC monolayers were exposed to benzalkonium chloride (BAK), sodium dodecyl sulfate (SDS), polyaminopropyl biguanide (PAPB), and ethylenediaminetetraacetic acid disodium salt (EDTA) for 5 min. Cell viability after exposure was then evaluated using PrestoBlue and alamarBlue. In set 3, HCEC monolayers were irradiated with UV radiation for 5min, 15min and 60min (the corresponding dose was 0.1719 J/cm2, 0.5156 J/cm2, and 2.0623 J/cm2, respectively). Then the cell viability was measured at 0h, 4h, and 24h after exposure using PrestoBlue.
Compared to the control without cells, both PrestoBlue and alamarBlue showed significantly higher fluorescent value for 5000 cells after 30 min incubation, and for 1000 cells after 1h, 2h, and 4h incubation (all p < 0.05). While the MTT assay only showed significantly higher absorbance for 5000 cells v.s the control without cells after 3h incubation (p < 0.05). PrestoBlue and alamarBlue also showed similar sensitivities in assessing the effect of various chemicals after 1h incubation, except that PrestoBlue was able to distinguish EDTA 0.01% and EDTA 0.02% from the medium control (p < 0.05), while alamarBlue was not. UV radiation caused dose-dependent decreases in cell viability in a time-dependent manner, with the highest cell viability at 0h (immediately after exposure); and the lowest at 24h after exposure.
Our study suggests that in evaluating the viability of HCEC, the PrestoBlue assay has the highest sensitivity at a shorter incubation time (1h), followed by the alamarBlue assay and the MTT assay, which indicates the PrestoBlue assay is a faster method. This study also demonstrates that the PrestoBlue assay can be used to evaluate the toxicity of UV radiation on HCEC cultures.
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