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A high‐throughput, fluorescence‐based cholesterol efflux assay (LB44)
Author(s) -
Saddar Sonika,
Kothambawala Tasnim,
Chacos Nick,
Saini Preeti,
Tchaga Grigoriy,
Yan Gordon
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.lb44
Subject(s) - efflux , reverse cholesterol transport , cholesterol , chemistry , cell , biochemistry , biology , lipoprotein
Numerous studies have established a negative correlation between cellular cholesterol efflux and cardiovascular disease.Cholesterol efflux from peripheral tissues and cells by a process called Reverse Cholesterol Transport (RCT). RCT allows extrahepatic cells, including macrophage‐derived foam cells in arterial atherosclerotic plaque, to transport excessive cholesterol back to the liver for bile acid synthesis and excretion, thus lowering the peripheral lipid burden. Standard protocols for quantitation of cholesterol efflux involve labeling cells with tritiated cholesterol and measuring the release of labeled sterol in a 12 or 24 well plate. This protocol, however is not ideal for high‐throughput screening of large numbers of serum samples or screening compounds/small molecules for their effect on cholesterol efflux. We report here a simple, convenient and high‐throughput cell‐based assay using fluorescently‐labeled cholesterol in a 96‐well plate format. Our results demonstrate that the percentage cholesterol efflux elicited by 2% ApoB‐depleted normal human serum in J774 macrophage cells is comparable to those published in literature. Our assay method utilizes a positive control for this assay which induces efflux independent of any receptor, that is used as a measure of the reliability of the assay. This assay is highly reproducible with low inter‐assay variability (蠄 10%). We demonstrate that this is an efficient method of measuring cholesterol efflux in a cell‐based assay. The increased sensitivity of the assay coupled with its high‐throughput screening capability enables scientists to screen large libraries of reagents and serum samples and provides a valuable tool for drug discovery in the field of cardiovascular disease research.