Effects of Mild and Severe Oxidative Stress on BACE1 Expression and APP Amyloidogenic Processing

Abstract This chapter describes methods for establishing oxidative stress conditions that do not induce cell death in a neuronal cell culture model. We termed these conditions "mild oxidative stress," as opposed to "severe oxidative stress," which results in significant cell loss. Mild oxidative stress resembles more closely what happens in the aging brain than severe oxidative stress. The protocols we have delineated include the preparation and maintenance of mouse primary cortical cultures, the induction of oxidative stress by treatment with hydrogen peroxide, the assessment of cell viability by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, the measurement of free radical production by the 2',7'-dichlorofluorescein (DCF) assay, and western blot analysis of the amyloid precursor protein (APP) and β-site APP cleaving enzyme, BACE1, two key proteins associated with Alzheimer's disease pathology and oxidative stress.