Intracellular Cholesterol and Mitochondrial GSH Analysis

Cholesterol levels were determined fluorometrically using the Amplex Red Cholesterol Assay kit (Thermo Fisher Sci.; A12216). Samples (0.5 × 10⁵ cells) were extracted with chloroform:isopropanol:IGEPAL CA-630 (7:11:0.1) and centrifuged at 13,000 g for 10 min to remove insoluble material. The organic phase was dried under vacuum, dissolved in 1 × cholesterol reaction buffer, and analyzed following the guidelines provided by the supplier. To evaluate the intracellular distribution of the cholesterol load, cells were fixed in 4% paraformaldehyde (PFA) for 20 min at room temperature, and after washing three times, were incubated with the naturally fluorescent polyene antibiotic filipin III (0.25 mg/ml; Sigma-Aldrich, F4767) for 30 min. Images were acquired with a Zeiss Axiophot fluorescence microscope using a 40 × /1.3 N.A. objective. The ImageJ software [34 (link)] was used to calculate the corrected total cell fluorescence (CTCF) by applying the following formula: CTCF = Integrated Density − (Area of selected cell × Mean fluorescence of background readings). Mitochondria from SH-SY5Y cells were isolated by digitonin fractionation as described previously [35 ] and the GSH content was analyzed using the Glutathione Assay Kit (Sigma-Aldrich, CS0260-1 KT) following the manufacturer's instructions.