Comparison of PrestoBlue and MTT assays of cellular viability in the assessment of anti-proliferative effects of plant extracts on human endothelial cells

Abstract

IntroductionPrestoBlue (PB) is a new, simple and extremely fast live assay to monitor cell viability and cytotoxicity.Herein, we compared two in vitro cytotoxicity assays, new (PB) and classic (MTT), in the assessment of viability of human umbilical vein endothelial cells (HUVECs) in the presence of selected plant extracts. MethodsThe anti-proliferative effects of two extracts from medicinal plants, i.e., walnut husk extract and spent hop extract, used at the concentration range of 1–200μg/ml of gallic acid equivalent, were compared with the effects recorded for resveratrol — a natural polyphenolic compound. Reduction of dyes by endothelial cells was determined colorimetrically (MTT and PB) and fluorometrically (PB). ResultsAt higher concentrations, all tested compounds caused significant loss of cell viability. Regardless of plant compound, the PB assay, when measured colorimetrically, produced higher EC50 values compared to other modes of measurement, however, the statistically significant differences in EC50 values among the assays were revealed only for spent hop extract. Conversely, the EC50 values for each plant compound obtained in MTT (colorimetric assay) and PB (fluorometric assay) were similar. According to EC50 values, the cytotoxicity of plant compounds ranked as follows: spent hop extract>resveratrol>walnut husk extract. Furthermore, the MTT assay showed overall lower inter-assay variability and higher signal-to-noise ratio compared to PB assay. DiscussionIn conclusion, we recommend fluorometric PrestoBlue assay for cytotoxicity assessment in human endothelial cells. Due to substantial differences in EC50 values and S/N ratios between spectrophotometric PB and MTT or fluorometric PB assays, colorimetric quantification of HUVECs' viability with the use of PB reagent should be avoided.