Description
Key facts
Detection method:Fluorescent,
Sample types:Purified protein, Adherent cells,
Assay type:Cell-based (quantitative),
Assay Platform:Microplate reader
Product details:
Cholesterol Efflux Assay Kit ab196985 is a high-throughput, cell-based screening assay for measuring cholesterol efflux capacity. The cholesterol efflux assay protocol uses fluorescently-labeled cholesterol (Ex/Em 485/523 nm).
It provides a safe, sensitive and reproducible method for measuring cholesterol efflux, avoiding the disadvantages of handling radioactive materials with the alternative commonly used triatiated cholesterol ([
H]cholesterol) efflux assay.
This cholesterol efflux assay can be used to:
- Screen small molecules for their effect on cholesterol efflux as a part of drug discovery program.
How the assay works
Cholesterol Efflux Assay ab196985 is an assay for measuring cholesterol efflux from cells and uptake by acceptor apoplipoproteins using a fluorescently-labeled cholesterol analogue.
Cells are incubated with a labeling reagent mix that inserts fluorescently-labelled cholesterol into the plasma membrane (exchanging unlabelled cholesterol for fluoresently-labelled cholesterol). The labelled cells are then treated with an equilibration reagent mix overnight. This prevents the esterification and breakdown of fluorophore-labeled cholesterol, allowing it to be recognized by ABC-family lipid transporters.
After washing, cholesterol efflux is initiated by the addition of sample containing cholesterol acceptor apoplipoproteins. Following 4 hours incubation, the supernatant (medium containing cholesterol acceptor) of each well is transferred to a white 96-well plate and the fluorescence (Ex/Em = 485/523 nm) is measured in endpoint mode. The the adherent cell monolayer is solubilized by adding lysis buffer and the lysate is transfered to another white 96-well microplate and fluorescence is measured.
Cholesterol efflux from the labeled macrophage cells to a particular cholesterol acceptor is calculated by dividing the fluorescence intensity (RFU) obtained for the supernatant by the sum of the fluorescence intensity of the supernatant and cell lysate of the same treatment.
The kit includes a reagent to remove apolipoprotein B-containing lipoprotein particles (LDL and VLDL) from serum samples, enabling quantification of the efflux/uptake capacity of serum HDL without potential interference from LDL.
Cholesterol efflux assay protocol summary
- Label cells with labeling and equilibration mix overnight
- Wash cells, treat as required and incubate
- Transfer cell supernatant to microplate, and separately solubilize cells with cell lysis buffer
- Analyze supernatant and cell lysates with microplate reader
Related Cholesterol assay products
Other cholesterol assay kits include:
- Cholesterol Assay Kit - HDL and LDL/VLDL
ab65390
- Cholesterol/ Cholesteryl Ester Assay Kit
ab65359
- Cell-based Cholesterol assay kit
ab133116
- Cholesterol Uptake assay kit
ab236212
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Background information
Cholesterol efflux from the peripheral tissues and cells in atherosclerotic plaque is an initial and critical step in Reverse Cholesterol Transport (RCT). RCT is the process by which extrahepatic cells, including macrophage-derived foam cells in arterial atherosclerotic plaque, export cholesterol to plasma high-density lipoprotein (HDL) particles via the action of transmembrane lipid transporters such as ABCA1 and ABCG1, following interaction of HDL particles with extracellular matrix proteins. This sequestered cholesterol is eventually transported from HDL to the liver for bile acid synthesis and excretion, thus lowering the peripheral lipid burden.
Other notes
This product is manufactured by BioVision, an Abcam company and was previously called K582 Cholesterol Efflux Fluorometric Assay Kit (cell-based). K582-100 is the same size as the 100 test size of ab196985.
The Safety Datasheet for this product has been updated for certain countries. Please check the current version in the Support and downloads section.
REACH authorisation
Abcam has not and does not intend to apply for the REACH Authorisation of customers' uses of products that contain European Authorisation list (Annex XIV) substances.
It is the responsibility of our customers to check the necessity of application of REACH Authorisation, and any other relevant authorisations, for their intended uses.
Properties and Storage Information:
Shipped at conditions-Blue Ice, Appropriate short-term storage conditions--20°C, Appropriate long-term storage conditions--20°C, Storage information--20°C
Supplementary Information:
This supplementary information is collated from multiple sources and compiled automatically.
Cholesterol efflux is a mechanical process where excess cholesterol is removed from cells and transported to extracellular acceptors. The process is managed by proteins like ATP-binding cassette transporter A1 (ABCA1) and ATP-binding cassette transporter G1 (ABCG1) which are sometimes referred to as key players in cholesterol efflux. These proteins have masses of about 220 kDa and 75 kDa respectively and are highly expressed in tissues such as the liver intestine and macrophages. They interact directly with lipid-poor apolipoproteins facilitating the removal of cellular cholesterol.
Biological function summary
Cholesterol efflux plays a significant role in maintaining lipid homeostasis and preventing lipid accumulation in cells. It is part of a larger complex known as the reverse cholesterol transport pathway which involves several lipoprotein particles including high-density lipoprotein (HDL). This pathway assists in transporting cholesterol from peripheral tissues back to the liver for excretion. By doing so it is instrumental in maintaining optimal cellular function and contributes to cardiovascular health.
Pathways
Mechanisms controlling cholesterol efflux are integrated into the broader framework of lipid metabolism and inflammation pathways. The first is the reverse cholesterol transport pathway as already mentioned which uses HDL to ferry cholesterol back to the liver. Proteins such as lecithin–cholesterol acyltransferase (LCAT) and lipid transfer proteins are also involved in these pathways facilitating the conversion of cholesterol to cholesteryl esters and subsequent transport. Cholesterol efflux also interacts with the inflammatory pathway where it connects with proteins like nuclear receptors that regulate inflammatory responses.
Imbalances in cholesterol efflux are linked to conditions such as atherosclerosis and Tangier disease. A deficiency in cholesterol efflux can lead tangibly to cholesterol buildup in blood vessels contributing to plaque formation and atherosclerosis. The transporter proteins ABCA1 and ABCG1 are connected to these conditions; for instance mutations in ABCA1 are directly related to Tangier disease which results in extremely low levels of HDL cholesterol. Understanding cholesterol efflux and its related mechanisms is essential for developing strategies to manage and treat related disorders.
