HDL MOA Assay: Efflux Assay Measured in Cell-Based System Using Plate Reader - 2085-06_Inhibitor_Dose_DryPowder_Activity_Set2
SR-BI also mediates bidirectional flux (e.g., efflux) of unesterified, or 'free' cholesterol (FC) between cells and HDL or other acceptors. In vivo, the greatest SR-BI-mediated selective uptake occurs in the liver and steroidogenic organs. This assay is used to determine if the compounds alter the efflux of free cholesterol to HDL. Compounds that do and do not inhibit efflux in a dose dependent manner will be of value. ..more
BioActive Compound: 1
Keywords: cholesterol efflux
SR-BI also mediates bidirectional flux (e.g., efflux) of unesterified, or 'free' cholesterol (FC) between cells and HDL or other acceptors. In vivo, the greatest SR-BI-mediated selective uptake occurs in the liver and steroidogenic organs. This assay is used to determine if the compounds alter the efflux of free cholesterol to HDL. Compounds that do and do not inhibit efflux in a dose dependent manner will be of value.
Expected Outcome: Some compounds will decrease efflux similar to BLT-1 and be labelled active in this assay. Compounds that increase or do not change efflux suggest a different mechanism from BLT-1. Any outcome is acceptable for a probe candidate but if efflux is inhibited, it must be at an IC50 of less than 10 uM.
For efflux assays, cells were seeded on day 0 on 24-well plates at a density of 50,000 cells per well in the same medium.
On day 1, the medium was replaced with Ham's F12 medium supplemented with 10% bovine lipoprotein deficient serum, 1 uCi/ml [1,2-3H]cholesterol (40-60 Ci/mmol; NEN Life Science), 2mML-glutamine, 50 U/ml penicillin, 50 mg/ml streptomycin, and, for ldlA[mSR-BI] cells only, 0.25 mg/ml G418.
(Hams+P/S+Gln+10% Serum + cholesterol +/-G418)
Cholesterol, [1,2-3H(N)]-, 1mCi (37MBq)
Product number: NET139001MC Perkin Elmer - NEN
On day 3, the cells were washed twice in 1 ml Ham's F12 medium without any supplements and then cultured for another 24 h in 1 ml tissue culture medium in which fetal bovine serum was replaced with 1% fatty acid-free (FAF) BSA (Sigma catalog No. A6003).
(Hams+P/S+Gln+1% FAF-BSA +/-G418)
Day 4: the efflux assay was performed on day 4. Cells were washed twice with Ham's F12 without supplements and then preincubated for 1 h at 37C with compounds at the indicated concentrations in 1 ml assay medium (Ham's F12 + P/S + Gln + 0.5% DMSO + 25 mM HEPES, pH 7.4+ 0.5% FAF BSA). Subsequently, the cells were incubated for an additional 2 h with the same concentrations of small molecules and with the indicated concentrations unlabeled HDL a 55 ul of assay medium containing 2 mg/ml HDL added to each well (final HDL concentration of 100 ug/ml)
(Ham's F12 + P/S + Gln + 0.5% DMSO + 25 mM HEPES, pH 7.4 + 0.5% FAF BSA)
After a 2-h incubation at 37 degrees C, the [3H]cholesterol contents of the cells and the media were determined as follows. 120 ul efflux media were collected and clarified by centrifugation for 1 min with a desktop microcentrifuge, and the radioactivity in 100 ul of each supernatant was determined by liquid scintillation counting (LSC). Cells were solubilized with 400 ul of lysis buffer (1% Triton X-100 in PBS) for 30 min at room temperature, and the amount of [3H]cholesterol in 100 ul of each lysate was determined by LSC. Total cellular [3H]cholesterol was calculated as the sum of the radioactivity in the efflux medium plus the radioactivity in the cells and was used to calculate the [3H]cholesterol efflux (percent of total [3H]cholesterol released into the medium).
Categorized Comment - additional comments and annotations
Assay Format: Cell-based
Assay Format: Cell-based
Assay Type: Functional
* Activity Concentration.
Data Table (Concise)