Cholesterol Efflux Assay with THP-1 Cells

A cholesterol efflux assay using THP-1 cells was carried out as described previously [12 (link)]. Briefly, THP-1 cells (2.5 × 10⁵ cells/well) were differentiated into macrophages via culture in RPMI-1640 medium containing 100 ng/ml of phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich) supplemented with 0.2% bovine serum albumin (BSA) for 2 days. Culture medium was then changed to RPMI-1640 containing acetylated LDLs (acLDLs) (50 μg of protein/ml), T0901317 (1 mmol/l; Enzo Life Sciences), a liver X receptor (LXR) agonist for promoting expression of ABCA1, ³H-cholesterol (1 μCi/ml; PerkinElmer), and BSA (0.2%). In doing so, the THP-1 macrophages were converted to foam cells. After equilibration with RPMI-1640 supplemented with T0901317 (1 mmol/l) and BSA (0.2%) for 18 h, the cells were incubated with BDS or HDL in RPMI-1640 for 4 h. CEC was calculated as the percentage of radioactivity in the medium as per the following formula: {³H-cholesterol in medium/(³H-cholesterol in medium + ³H-cholesterol in cells)} × 100 − the percentage of passive diffusion in the case of no cholesterol acceptor. All samples were assayed in triplicate.