2.9. Measurement of HDL capacity to reduce macrophage cholesterol

To measure the capacity of HDL to reduce macrophage cholesterol content, an enzymatic cholesterol assay was used as described [31,32]. Briefly, Apoe^−/− macrophages were incubated for 48 h with DMEM containing acetylated LDL (Alfa aesar, cat#: J65029, 40 μg/ml). The cells were then washed and incubated for 1 h in DMEM containing 0.1% BSA. The cells were then washed and incubated for 24 h in DMEM supplemented with HEPES in the presence of 50 μg/mL human HDL, modified HDL, or 2.5% apoB-depleted serum from mice. Cellular cholesterol was measured after incubation with DMEM alone or with cholesterol acceptor using an enzymatic cholesterol assay [31,32]. Briefly, isopropanol/NP-40 (vol/vol:9/1) was added to the dried isopropanol lipid extracts and vortexed for 10 s. Then 40 ul of each sample was added to 96-well plates and catalase solutions (Sigma, cat#:C1345) in 50 mM potassium phosphate buffer were added to the wells for both standards and samples to lower the background. Then, 100 ul of the enzyme and fluorescent probe mixture (ADHP, cat#: AnaSpec # 85,500; cholesterol oxidase, Cat#: C8649–100U; cholesterol esterase, Cat#:C9281–100UN; horseradish peroxidase, Cat#: P8375-1kun) were prepared in reagent A buffer (sodium chloride, cholic acid, Triton-100 in 100 mM potassium phosphate buffer) and added into each well. The fluorescence intensity (excitation wavelength 530 nm, emission wavelength 580 nm) was measured immediately after incubation at 37 °C for 25 min s. The cholesterol efflux capacity was calculated as ((cellular cholesterol with DMEM alone - cellular cholesterol with acceptor)/cellular cholesterol with DMEM alone ∗ 100).