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BioAssay: AID 624017

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
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 Related BioAssays
 Related BioAssays
AID: 624017
Data Source: Broad Institute (2085-06_Inhibitor_Dose_DryPowder_Activity_Set2)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2012-04-05
Hold-until Date: 2013-03-19
Modify Date: 2013-03-19

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compound: 1
Related Experiments
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AIDNameTypeComment
488952Broad Institute Inihibitors of the HDL Receptor, SR-BI Inhibitors Probe ProjectSummarydepositor-specified cross reference: Summary assay
488896HTS using DiI-HDL to assay lipid transfer in ldlA[SR-BI] cells Measured in Cell-Based System Using Plate Reader - 2085-01_Inhibitor_SinglePoint_HTS_ActivityScreeningsame project related to Summary assay
493194HTS using DiI-HDL to assay lipid transfer in ldlA[SR-BI] cells Measured in Cell-Based System Using Plate Reader - 2085-01_Inhibitor_Dose_CherryPick_ActivityConfirmatorysame project related to Summary assay
540246HDL Counter Assay: 3/24 Cytotox Measured in Cell-Based System Using Plate Reader - 2085-02_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
540326DiI Quencher Assay Measured in Biochemical System Using Plate Reader - 2085-08_Inhibitor_Dose_CherryPick_ActivityConfirmatorysame project related to Summary assay
540354HTS using DiI-HDL to assay lipid transfer in ldlA[SR-BI] cells Measured in Cell-Based System Using Plate Reader - 2085-01_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
588392HTS using DiI-HDL to assay lipid transfer in ldlA[SR-BI] cells Measured in Cell-Based System Using Plate Reader - 2085-01_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
588403DiI Quencher Assay Measured in Biochemical System Using Plate Reader - 2085-08_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
588548HTS using DiI-HDL to assay lipid transfer in ldlA[SR-BI] cells Measured in Cell-Based System Using Plate Reader - 2085-01_Inhibitor_Dose_DryPowder_Activity_Set4Confirmatorysame project related to Summary assay
588553HTS using DiI-HDL to assay lipid transfer in ldlA[SR-BI] cells Measured in Cell-Based System Using Plate Reader - 2085-01_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatorysame project related to Summary assay
588754HTS using DiI-HDL to assay lipid transfer in ldlA[SR-BI] cells Measured in Cell-Based System Using Plate Reader - 2085-01_Inhibitor_Dose_DryPowder_Activity_Set5Confirmatorysame project related to Summary assay
588777HDL Orthogonal Assay: Alexa 488-HDL Measured in Cell-Based System Using Plate Reader - 2085-03_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
588810HDL Orthogonal Assay: Alexa 488-HDL Measured in Cell-Based System Using Plate Reader - 2085-03_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
588818HDL MOA Assay: Efflux Assay Measured in Cell-Based System Using Plate Reader - 2085-06_Inhibitor_SinglePoint_DryPowder_ActivityOthersame project related to Summary assay
588825HDL Uptake counterscreen Measured in Cell-Based System Using Plate Reader - 2085-07_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
588826HDL Counter Assay: 3/24 Cytotox Measured in Cell-Based System Using Plate Reader - 2085-02_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatorysame project related to Summary assay
588828HTS using DiI-HDL to assay lipid transfer in ldlA[SR-BI] cells Measured in Cell-Based System Using Plate Reader - 2085-01_Inhibitor_Dose_DryPowder_Activity_Set6Confirmatorysame project related to Summary assay
588829HDL Counter Assay: 3/24 Cytotox Measured in Cell-Based System Using Plate Reader - 2085-02_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
588830HDL Counter Assay: 3/24 Cytotox Measured in Cell-Based System Using Plate Reader - 2085-11_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
588831HDL Inhib: Reversibility Measured in Cell-Based System Using Plate Reader - 2085-14_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
588833HTS using DiI-HDL to assay lipid transfer in ldlA[SR-BI] cells Measured in Cell-Based System Using Plate Reader - 2085-01_Inhibitor_Dose_DryPowder_Activity_Set7Confirmatorysame project related to Summary assay
588836HDL Inhib: Activity in wild type cell line Measured in Cell-Based System Using Scintillation - 2085-12_Inhibitor_SinglePoint_DryPowder_ActivityOthersame project related to Summary assay
588837HDL Inhib: Activity in mutant cell line Measured in Cell-Based System Using Scintillation - 2085-13_Inhibitor_SinglePoint_DryPowder_ActivityOthersame project related to Summary assay
588842HDL Counter Assay: 3/24 Cytotox Measured in Cell-Based System Using Plate Reader - 2085-02_Inhibitor_Dose_DryPowder_Activity_Set4Confirmatorysame project related to Summary assay
588843HDL Inhib: Activity in mutant cell line Measured in Cell-Based System Using Plate Reader - 2085-10_Inhibitor_SinglePoint_DryPowder_ActivityOthersame project related to Summary assay
602126HDL Counter Assay: 3/24 Cytotox Measured in Cell-Based System Using Plate Reader - 2085-02_Inhibitor_Dose_DryPowder_Activity_Set5Confirmatorysame project related to Summary assay
602134HDL Counter Assay: Tf Endo Assay Measured in Cell-Based System Using Plate Reader - 2085-04_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
602138HDL Inhib: Activity in mutant cell line Measured in Cell-Based System Using Plate Reader - 2085-10_Inhibitor_Dose_DryPowder_ActivityOthersame project related to Summary assay
602152HDL MOA Assay: Efflux Assay Measured in Cell-Based System Using Plate Reader - 2085-06_Inhibitor_Dose_DryPowder_ActivityOthersame project related to Summary assay
602154HDL Inhib: Reversibility Measured in Cell-Based System Using Plate Reader - 2085-15_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
602155Liposome Uptake Assay Measured in Biochemical System Using Scintillation - 2085-16_Inhibitor_Dose_DryPowder_ActivityOthersame project related to Summary assay
602285HTS using DiI-HDL to assay lipid transfer in ldlA[SR-BI] cells Measured in Cell-Based System Using Plate Reader - 2085-01_Inhibitor_Dose_DryPowder_Activity_Set8Confirmatorysame project related to Summary assay
623880HTS using DiI-HDL to assay lipid transfer in ldlA[SR-BI] cells Measured in Cell-Based System Using Plate Reader - 2085-01_Inhibitor_Dose_DryPowder_Activity_Set9Confirmatorysame project related to Summary assay
623895HDL Counter Assay: 3/24 Cytotox Measured in Cell-Based System Using Plate Reader - 2085-02_Inhibitor_Dose_DryPowder_Activity_Set6Confirmatorysame project related to Summary assay
624007HDL Uptake counterscreen Measured in Cell-Based System Using Plate Reader - 2085-07_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
624010HDL Inhib: Activity in mutant cell line Measured in Cell-Based System Using Scintillation - 2085-13_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
624015HDL Inhib: Activity in mutant cell line Measured in Cell-Based System Using Plate Reader - 2085-10_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
624022HDL Inhib: Activity in wild type cell line Measured in Cell-Based System Using Scintillation - 2085-12_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
624024HDL Orthogonal Assay: Alexa 488-HDL Measured in Cell-Based System Using Plate Reader - 2085-03_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatorysame project related to Summary assay
624026HDL Counter Assay: 3/24 Cytotox Measured in Cell-Based System Using Plate Reader - 2085-02_Inhibitor_Dose_DryPowder_Activity_Set7Confirmatorysame project related to Summary assay
624029HTS using DiI-HDL to assay lipid transfer in ldlA[SR-BI] cells Measured in Cell-Based System Using Plate Reader - 2085-01_Inhibitor_Dose_DryPowder_Activity_Set10Confirmatorysame project related to Summary assay
Description:
Keywords: cholesterol efflux


Assay Overview:
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.
Protocol
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).
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1AC50_uM *FloatμM

* Activity Concentration.
Additional Information
Grant Number: 2 R01 HL052212-11

Data Table (Concise)
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