ab133116 includes filipin III, fixative, and wash buffer in a ready to  use format. It provides a simple fluorometric method to study mechanisms and biological factors that regulate cholesterol  metabolism or movement within cells. A cholesterol trafficking  inhibitor, U-18666A, is included as a positive control. THP-1 cells are  differentiated by 100 ng/ml of phorbol and treat with Hoechst 33258 stain and the caspase.After lipid loading, microscopy is used to examine the subcellular localization of the accumulated lipids as well as to analyze changes in lysosomal environment. LysoSensor Yellow/Blue DND-160 staining (Molecular Probes, Eugene, THP-1 macrophages  are  plated onto 35 mm wells or coverslips at a density of 1.5 × 106 cells and incubated for 3–4 days at 37°C in RPMI containing 10% FBS and 50 ng/ml phorbol ester (TPA) to allow for differentiation into macrophages. Culture media for all incubations is supplemented with HEPES (20 mmol/l), Eagle’s vitamins, l-glutamine (200 mmol/l), streptomycin (100 µg/ml), penicillin (100 IU/ml), and β-mercaptoethanol (0.008 µl/ml). TPA is included in the incubation medium throughout the duration of the experiments. Macrophages  are  incubated with medium containing 1% FA-free BSA for 24 h before cholesterol loading to minimize excess TG in cells prior to lipid loading.

.After lipid loading, microscopy is used to examine the subcellular localization of the accumulated lipids as well as to analyze changes in lysosomal environment. LysoSensor Yellow/Blue DND-160 staining (Molecular Probes, Eugene, OR) is used to determine changes in lysosomal pH (44, 45). This dye fluoresces yellow in an acidic environment, but the fluorescence wavelength shifts toward blue as the environment becomes more alkaline. For staining, cells should wash two times in PBS, and the dye was add to cells at a concentration of 5 µM in medium containing 1% FBS. All images should collect within 10 min after the placement of dye on the cells to avoid artifacts produced by the alkaline properties of the dye.

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