Cellular cholesterol efflux assays

RAW264.7 macrophages were seeded into 6-well plates (1×10⁶ cells/well), radiolabeled with 5 μCi/ml [³H]-cholesterol and loaded with 100 μg/ml ac-LDL for 48 h in media containing 0.2% BSA. Afterward, equilibrated [³H]-cholesterol-labeled cells were washed with fresh media and subsequently treated as indicated in the figures. Then, the cells were rinsed again with PBS and incubated in the presence of apoA-1 (10 μg/ml) or HDL (50 μg/ml) for 24 h. Medium and cell-associated [³H]-cholesterol were then determined by LSC. Finally, the percent efflux was calculated using the following equation: [total media counts / (total cellular counts + total media counts)] × 100%.