Cholesterol efflux to total plasma and apoB-depleted human plasma were determined using the human monocyte cell line THP-1 as cholesterol donor. THP-1 cells were obtained from European Collection of Cell Cultures and maintained in medium A (RPMI 1640 with 25 mmol/L HEPES buffer, supplemented with 10% fetal bovine serum, 1% l-glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin) at 37°C in 5% CO2. Before the experiment, THP-1 cells were seeded into 24-well plates at density of 5 × 105 cells per well and differentiated into macrophages with 0.1 μmol/L phorbol 12-myristate-13-acetate (no. P1585; Sigma Aldrich) within 3 days. Macrophages were washed three times with PBS and incubated in medium B (RPMI 1640 with 25 mmol/L HEPES buffer, supplemented with 2% fetal bovine serum, 1% l-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin, 50 μg/mL acetyl-LDL, and 10 μCi/mL [1α,2α(n)-3H]-cholesterol (no. NET139001MC; Perkin Elmer, the Netherlands) for 1 day at 37°C in 5% CO2. After incubation, cells were washed three times with PBS and the efflux assay was started by adding total human plasma or apoB-depleted human plasma diluted to 1% in medium C (RPMI 1640 with 25 mmol/L HEPES buffer, supplemented with 1% l-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin, and 0.5 mg/mL BSA). The whole assay was carried out in triplicate. To be able to normalize results between series of experiments and to correct for plate-to-plate variation, efflux to a standard preparation of HDL (50 μg protein/mL) was determined in triplicate. After 4-h incubation, medium was collected and centrifuged. Subsequently, [3H]cholesterol was quantified by liquid scintillation counting. Total cellular 3H-cholesterol was determined after extraction of the cells with 0.1 mol/L NaOH. Cholesterol efflux rate was calculated by dividing the 3H activity in the medium by the sum of the 3H activity in the medium and the cell extract. Background values (the efflux in the absence of plasma) were subtracted.