Figure 1.

Principle of the cholesterol efflux assay. J774 macrophages are cultivated in multiwell plates to form a monolayer. The cells are then treated for 24 h with an ACAT (acyl coenzyme A: cholesterol acyltransferase) inhibitor and radiolabeled cholesterol ([³H]-cholesterol). The ACAT inhibitor prevents cholesterol esterification and the added cholesterol remains cell-associated as free cholesterol. On the following day, the cells are treated with cyclic adenosine monophosphate (cAMP) for 16 h to stimulate the expression of the cholesterol exporter ABCA1. The cholesterol efflux in unstimulated macrophages is mediated to 15% by ABCA1, 25% by SR-BI and 55% by passive diffusion (includes ABCG1-mediated efflux). By cAMP treatment, the ABCA1-dependent cholesterol efflux triples to about 40%, while passive diffusion accounts for 50% and SR-BI-mediated efflux for 10% [125]. Human serum shows a depletion of lipoproteins containing apoB100 (mainly VLDL, LDL) using polyethylene glycol. After extensive rinsing of the cells, apoB-depleted serum (containing all HDL subclasses) is added to the [³H]-cholesterol-labeled macrophages at a concentration of 2.8%. After 4 h, the [³H]-cholesterol that has passed from the cells into the supernatant is quantified by liquid scintillation counting.