Both oxidative stress-dependent and independent effects of amyloid β protein are detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay

Abstract

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay has been widely used for evaluating amyloid β protein (Aβ) toxicity. However, the potency of Aβ in inhibiting cellular MTT reduction and the underlying mechanism have been reported with some discrepancies among researchers. To understand what makes such discrepancies, the effect of Aβ detected by MTT reduction assay was re-examined in detail by using cultured rat hippocampal neurons. Micromolar concentrations (>10 μM) of Aβ caused a decrease in cell viability, which resulted in a decrease in MTT reduction per well regardless of assay time. The micromolar Aβ-induced decrease of cellular MTT reduction was significantly attenuated by antioxidants (catalase, propyl gallate or Trolox). On the other hand, nanomolar Aβ did not affect cellular MTT reduction activity at an initial stage of assay (<1 h), and decreased the total production of MTT formazan by accelerating the exocytosis of MTT formazan when MTT assay was performed for a longer time (>2 h). The assay time-dependent, nanomolar Aβ-induced decrease of cellular MTT reduction was not at all affected by antioxidants. Furthermore, subtoxic concentration of H 2O 2 failed to mimic the effect of nanomolar Aβ on MTT reduction. These results indicate that micromolar Aβ-induced, oxidative cell death is detected by MTT assay regardless of assay time, whereas nanomolar Aβ-induced acceleration of MTT formazan exocytosis is not mediated by oxidative stress and detected only when MTT assay is performed for a longer time. The time of MTT assay should be properly chosen depending on the purpose of the study.