Amplex Red Assay

The cells and brain tissue were lysed with lysis buffer (50 mM phosphate buffer, 500 mM NaCl, 25 mM cholic acid, and 0.5% Triron X-100). The unesterified cholesterol was determined using the Amplex Red Cholesterol Assay Kit (Molecular Probes) according to the manufacturer’s instructions. After incubation for 30 min at 37°C, the fluorescence intensities were measured on a fluorescence microplate reader (Molecular Devices) equipped with a filter set for excitation and emission at 560 ± 10 nm and 590 ± 10 nm, respectively. The cholesterol content was calculated with a cholesterol standard curve. Cellular cholesterol content was normalized to protein content. Data were collected from 4 to 6 mice per group. Randomization procedures are not applicable to these experiments. G-power software was used for sample size estimation. No data were excluded.