title: HDL-Mediated Cellular Cholesterol Efflux Assay Method.
abstract: Biomarkers of high-density lipoprotein (HDL) function may provide mechanistic insights and better cardiovascular risk discrimination than HDL-cholesterol mass. The purpose of this work is to describe a simplified experimental protocol that can be used in the determination of cholesterol efflux from macrophages cultured cells and be brought to a medium throughput volume. The cellular cholesterol efflux assay is designed to quantify the rate of cholesterol efflux from cultured cells to an acceptor particle or to plasma. This assay is multi step, cell based assay. Various factors, if not carefully controlled may influence the accuracy and reproducibility of the assay. Attempts were made to address factors influencing this assay and to provide a standardized method that is relatively rapid and scalable. We demonstrate that further centrifugation of the HDL fraction is necessary to avoid apolipoprotein B contamination when using polyethylene glycol (PEG) method. We demonstrate also no effect on cholesterol efflux efficiency when using PEG with plasma or serum. This method has been previously applied in our laboratory in context of cardiovascular research, cardiovascular disease and pharmacologic therapies.
authors.affiliation: Cardiovascular Research Laboratories, Research Institute of the McGill University Health Center, Montreal, Quebec, Canada anouar.hafiane@mail.mcgill.ca anouarhafiane@yahoo.com.
authors.forename: Anouar
authors.initials: A
authors.lastname: Hafiane
authors.affiliation: Cardiovascular Research Laboratories, Research Institute of the McGill University Health Center, Montreal, Quebec, Canada.
authors.forename: Jacques
authors.initials: J
authors.lastname: Genest
mesh_terms: D064286:ATP Binding Cassette Transporter 1 / Q000097:blood; D000818:Animals; D002460:Cell Line; D002498:Centrifugation; D002784:Cholesterol / Q000032:analysis / Q000097:blood*; D005260:Female; D006403:Hematologic Tests / Q000379:methods*; D006801:Humans; D008075:Lipoproteins, HDL / Q000097:blood* / Q000737:chemistry; D008264:Macrophages / Q000378:metabolism; D008297:Male; D051379:Mice; D011092:Polyethylene Glycols / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: C577836:ABCA1 protein, human; D064286:ATP Binding Cassette Transporter 1; D008075:Lipoproteins, HDL; D011092:Polyethylene Glycols; D002784:Cholesterol
keywords: ABCA1; Apolipoprotein A-I; Cholesterol efflux; HDL; Reverse cholesterol transport; polyethylene glycol