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
([3H]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:
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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.
