Primary cerebellar cells were plated into 96-well poly-l -lysine–coated black plates (Costar) and cultured for one (neurons) or seven (astrocytes) days prior to overnight incubation with 100 μM d -α-tocopherol (Acros Organics). The following day, the cells were incubated in 10 μg/ml 2',7'-dichlorodihydrofluorescein diacetate in HBSS without phenol red (Invitrogen) for 1 h, washed, and challenged with the indicated concentration of H2O2 for 1 h. After a final wash in HBSS, DCF fluorescence was measured on a PerkinElmer Victor 3 plate reader (excitation = 485 nm; emission= 535 nm) and normalized to DNA content as measured after incubating cells with bisbenzamide (Sigma; 2.5 μg/ml in 2 M NaCl, 50 mM Na2HPO4 [pH 7.4] in the dark at 37 °C for 60 min) in a plate reader (excitation = 365 nm; emission = 460 nm).
Statistical analyses and graphing were done using GraphPad Prism 6 (GraphPad Software, Inc). Student's t test and one-way ANOVA with Tukey's multiple comparisons post hoc analysis were performed, and threshold for statistical significance was set at p < 0.05.
Statistical analyses and graphing were done using GraphPad Prism 6 (GraphPad Software, Inc). Student's t test and one-way ANOVA with Tukey's multiple comparisons post hoc analysis were performed, and threshold for statistical significance was set at p < 0.05.