Cholesterol Efflux Assays

PMA-differentiated human THP1 macrophages, a physiological relevant human cell line in which cholesterol efflux is mediated by all known pathways relevant to macrophages, were used to determine CEC as described before.21 In brief, THP1 macrophages were loaded with cholesterol using acetylated human LDL (25 μg/ml) and labeled with [³H]-cholesterol (1 μCi/ml) for 48 hours. To maintain relevance to the physiological conditions (i.e., storage of cholesterol in macrophage foam cells as cholesteryl esters or CE and not FC), no acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor was included. After a 24-hour equilibration in serum-free medium, FC efflux was initiated by the addition of 1% serum or cholesterol acceptors at physiological concentrations present in 1% serum (albumin 0.5 mg/ml or HDL 150 μg/ml). Conditioned medium was collected after 4 hours and percent FC efflux was calculated as described.21 When assessing CEC of serum samples from human subjects, a control pooled serum sample was included while performing these cholesterol efflux assays and CEC of serum samples from human subjects was normalized to the percent efflux obtained using this control serum sample and expressed as a percentage.