2.6. HDL Cholesterol Efflux Capacity Assay

The cholesterol efflux capacity of HDL was determined in cholesterol-loaded murine macrophages as previously reported [26,27]. To this end, J774A.1 mouse macrophages were cultured in RPMI 1640 (Roswell Park Memorial Institute medium) containing 10% of heat-inactivated FBS (Fetal bovine serum), 2 mM glutamine, 100 U/mL penicillin, 100 U/mL streptomycin and 10 μg/mL gentamicin at 37 °C in a humidified atmosphere of 5% CO₂.

For the experiments, macrophages (1.5 × 10⁵ cells/well seeded in 6-well culture plates) were labeled for 48 h with [1α, 2α (n)⁻³H cholesterol] (GE Healthcare, Chicago, IL, USA) at 1 μCi per well. Cells were equilibrated overnight in 0.2% bovine serum albumin (BSA) and thereafter incubated with RPMI media containing 5% apoB-depleted serum (4 h, 37 °C) to promote cholesterol efflux from the [³H] cholesterol-labeled cells. Radioactivity signal was quantified in both media and cells and the percentages of cholesterol efflux calculated by expressing the radioactive cholesterol released to the medium as the fraction (%) of the total radioactive cholesterol present in the well (radioactivity in the cell + radioactivity in medium).