Cell cholesterol efflux to purified acceptors

Cell cholesterol efflux assays were conducted as previously described [21]. SH-SY5Y, N9, A172, and J774 cells were seeded in 24-well plates at 1/10 dilution from confluent tissue culture flasks, allowed to grow for 24h in the maintenance medium/10% FBS and incubated with 2μCi/mL [1,2-³H(N)]cholesterol (Perkin Elmer) in DMEM/2.5% FBS or RPMI/2.5% FBS, depending on the cell type (see above), for 24h. The human SH-SY5Y and A172 cells were then treated with 2μM liver X receptor (LXR) agonist T0901317 (Tocris Bioscience) or corresponding vehicle, while the mouse N9 and J774 cells were treated with 0.3mM 8-(4-chlorophenylthio)adenosine 3’,5’-cyclic monophosphate (8-CPT-cAMP; Sigma-Aldrich) or corresponding vehicle, in FBS-free cell type-appropriate medium/0.2% BSA for 18h. To measure efflux, the cells were exposed to FBS-free cell type-appropriate medium/± 2μM T0901317 or ± 0.3mM 8-CPTcAMP containing 10g/mL apolipoprotein A-I (apo A-I) or 50g/mL HDL or lacking cholesterol acceptors for 4h. Efflux medium was filtered using 0.45μm pore-size filter plates to eliminate floating cells. Cell lipids were extracted with hexane/isopropanol (3:2, v/v); the solvent was evaporated. The cell extracts and aliquots of the efflux media were read in a scintillation counter. Cholesterol efflux was expressed as the percentage of [³H]cholesterol counts in the efflux medium from the total of [³H]cholesterol counts in the medium and cells. Apo A-I and HDL were purified from human apheresis plasma.