ABCA1‐Mediated Cholesterol Efflux

The protocol to measure ABCA1‐mediated cholesterol efflux was published25 and modified for using CSF as the cholesterol acceptor. Briefly, BHK cell lines transfected with a mifepristone switch to express ABCA1 was used.26 BHK cell lines were plated at 20 000 per well with high‐glucose DMEM plus 10% FBS in 96‐well plates. On the second day, cells were labeled with 1 μCi/mL (³H) cholesterol (Moravek) using serum‐free high‐glucose DMEM, 2 mg/mL fatty acid–free albumin (Sigma‐Aldrich), and 2 μg/mL acyl‐coenzyme A:cholesterol acyltransferase inhibitor (Sigma‐Aldrich). On the third day, ABCA1 gene expression was induced with 10 nmol/L mifepristone overnight. On the following day, cholesterol acceptors or media alone were added to each well, with efflux assessed by the ratio of cholesterol in the media by total cholesterol (cells and media) 4 hours after incubation with acceptor or media. To test the efficiency of ABCA1 expression, purified apoE and apoA‐1 (Academy Biomedical) were used in increasing doses as cholesterol acceptors with and without mifepristone. As demonstrated in Figure S1A, apoA‐I and apoE efficiently accepted labeled (³H) cholesterol and did so in a dose‐dependent manner (Figure S1B). Control CSF between 0% and 100% of media was used to optimize the concentration of the CSF used in this assay. CSF was an efficient acceptor of cholesterol, and increasing CSF volume from 25 to 100 μL demonstrated a linear cholesterol efflux response (Figure S1C). Cholesterol efflux from the individual samples was then assessed using 35 μL CSF per well. Samples were run in triplicate, and the intraplate coefficient of variation for CSF samples was <10%. Furthermore, 5 μg/mL of purified apoE was run as a control in every plate. The interplate coefficient of variation was 11%.