4.7. Cholesterol Efflux Assay

Macrophage-specific cholesterol efflux capacity was measured using a commercially available Cholesterol Efflux Assay Kit (Abcam, ab196985, Cambridge, MA, USA). Briefly, THP-1-derived macrophages (1 × 10⁵) were labeled with a labeling reagent which includes a fluorescent labelled cholesterol and equilibrated for 24 h in a humidified incubator at 37 °C and 5% CO₂. Then, the media was removed, and cells were washed with fresh media and treated with RA (1, 10, 50 and 100 µM) for 24 h. Cells were added with HDL or ApoA1 cholesterol acceptors and incubated for 6 h more. Thereafter, the cholesterol contents in media and cell lysis were measured and cholesterol efflux to HDL or ApoA1 was calculated as follows: