2.5. Cholesterol efflux assay

In order to determine whether changes in the HDL‐bound proteins of interest are associated with changes in the HDL function, we measured the CEC of HDL as previously described by our group. ³ In summary, the HDL‐CEC assays were performed using the murine macrophage cell line, J774, as per published methods. ^{17 , 18 , 19} Briefly, 3 x 105 J774 cells/well were plated and radiolabeled for 24 h with 2 µCi of ³H‐cholesterol/ml. ATP‐binding cassette transporter A1 (ABCA1) was up‐regulated by incubation with 0.3 mmol/L 8‐(4‐chlorophenylthio)‐cAMP for 16‐hours. ApoB‐depleted serum (2.8%) from participants was added to the efflux medium for 4 h. Liquid scintillation counting was then added to quantify the efflux of radioactive cholesterol from the cells. Efflux was calculated using the standard formula: (µCi of ³H‐cholesterol in media containing 2.8% apoB‐depleted participant plasma‐µCi of ³H‐cholesterol in plasma‐free media / µCi of ³H‐cholesterol in media containing 2.8% apoB‐depleted pooled control plasma‐µCi of ³H‐cholesterol in pooled control plasma‐free media). The pooled plasma control was obtained from five healthy adult volunteers and all assays were performed in duplicates run at the same time.