Cholesterol efflux assay

Cholesterol efflux assay was performed following the manual instruction (Biovision, CA, USA). In brief, RAW264.7 macrophages were plated at the density of 1 × 10⁵ cells/well in a 96-well plate and maintained in DMEM plus 10 % FBS (Sigma-Aldrich) for 2 h. The adherent cells were incubated with labeled cholesterol for 16 h and then exposed to 100 μg/ml HDLs for 4 h. The supernatant were transferred to a 96-well plate to measure the fluorescence (Ex/Em = 482/515 nm). The adherent cells were solubilized by cell lysis buffer to measure the fluorescence (Ex/Em = 482/515 nm). Cholesterol efflux % = fluorescence intensity of the media/(fluorescence intensity of the cell lysate + media) × 100 [42].