Cholesterol Efflux Assay

RAW cells were grown to 60% confluence in 24-well plates, and then incubated with DMEM containing 1 μCi of [1, 2-³H] cholesterol per ml for 48 h. RAW cells were washed three times with PBS, and Cellular cholesterol pools were allowed to equilibrate for another 24 h in DMEM containing 0.1% BSA (DMEM-BSA), Ad-GFP or Ad-shNAMPT (10 μl 1 × 10¹¹ pfu/ml). For dose-dependent experiments, RAW cells were stimulated with recombinant mouse NAMPT (Alexis Biochemicals, Paris, France) in concentrations of (40, 100 and 200 nM) for another 24 h. After intensive washing of cells with PBS-BSA, Efflux studies (24 h) were carried out using 100 μg of HDL per ml prepared in DMEM-BSA as the cholesterol acceptor. After the efflux period, Medium was collected and centrifuged, and radioactivity was counted by liquid scintillation counting (LSC). The residual radioactivity in the cell fraction was determined after an overnight extraction with hexane-isopropanol (3:2). Percentages of cholesterol efflux to HDL were calculated by dividing the radioactive counts in the efflux medium by the sum of the radioactive counts in the medium and the cell fraction. The values were normalized with untreated control cells expressed as 100%.