Cholesterol efflux assay

Bodipy cholesterol (2 μg/ml, Avanti Polar Lipids Inc., Alabaster, AL, USA) was added to cells during the 16-h initial treatment period of the 27OH supplementation experiment. Unlabeled cells were used to control for background fluorescence. Media was aspirated and cells were washed once with PBS, and then incubated 4 h in treatment medium as described earlier. Media were collected and cells were lysed using M-PER reagent (Fisher Scientific). The fluorescence from cells and media was quantified, and cholesterol efflux was calculated by the equation 100 × (media fluorescence)/(cell lysate fluorescence + media fluorescence).