Cholesterol Efflux Assay Protocol

The cholesterol efflux assay was performed as earlier [17 (link)]. The experimental protocol is shown schematically in Fig. 1. Fibroblasts were seeded into 12-well cell culture plates and grown to 50% confluence in FSF growth medium prior to addition of either 1 μCi/mL or 0.5 μCi/mL ([1α,2α(n)-³H]cholesterol (Amersham Biosciences). The cells were allowed to reach confluence, the radioactive medium was aspirated, the cells were washed twice and incubated with 1 mL of DMEM/BSA containing 20 μg/mL of cholesterol with or without 100 μg/mL PC. After 24 hr, the medium was aspirated and the cells were incubated in DMEM/BSA containing 0.5 mM 8-Br-cAMP with or without 12.5 μg/mL apolipoprotein E for 24 hours. The plates were chilled on ice, the medium was collected into scintillation vials and the cells were washed 4 times with cold (4°C) HEPES-saline BSA (0.03 mM BSA, 138mM NaCl, 5.7 mM KCl, 1.8 mM CaCl₂, 0.8mM MgSO₄, 3.7 mM HEPES, pH 7.4) to remove adherent lipids. A 3:2 hexanes: Isopropanol (v/v) solvent was used to extract cellular lipids [18 (link)]. Radioactivity was measured using complete counting cocktail (3a70B, Research Products International) and a Beckman, LS 6500 scintillation counter. The amount of the effluxed cholesterol was calculated as: Cholesterol efflux (%) = [radioactivity in the medium/(radioactivity in the medium + radioactivity in the lipid extract)] × 100.