Measuring cholesterol efflux involves the tracking of cholesterol movement out of cells. Cholesterol efflux is an essential mechanism to maintain cellular cholesterol homeostasis, and this process is largely regulated via the LXR transcription factors and their regulated genes, the ATP-binding cassette (ABC) cholesterol transporters ABCA1 and ABCG1. Typically, efflux assays are performed utilizing radiolabeled cholesterol tracers to label intracellular cholesterol pools, and these assays may be tailored to quantify the efflux of exogenously delivered cholesterol or alternatively the efflux of newly synthesized (endogenous) cholesterol, in different cell types (macrophages, hepatocytes). Cholesterol efflux may also be customized to quantify cholesterol flux out of the cell to various exogenous cholesterol acceptors, such as apolipoprotein A-I, high-density lipoprotein, or methyl-beta-cyclodextrin, depending on the purpose of the experiment. Here, we provide comprehensive protocols to quantify the net flux of cholesterol out of cells and recommendations on how this assay may be tailored as a function of the experimental question at hand.
Keywords: ABCA1; ABCG1; ApoA-I; Cholesterol mass assay; Efflux; Endogenous cholesterol; Exogenous cholesterol; Fluorescent cholesterol efflux; HDL; Methyl-beta-cyclodextrin; Phospholipid.