Bookmark and Share
BioAssay: AID 588836

HDL Inhib: Activity in wild type cell line Measured in Cell-Based System Using Scintillation - 2085-12_Inhibitor_SinglePoint_DryPowder_Activity

The goal of this assay is to verify compounds that disrupt the binding of HDL particles to SR-BI scavenger receptor using an alternative means of labeling HDL-cholesterol particles and avoiding any type of fluorescence measurement. To measure this binding event, HDL particles are labeled with the[H3}-cholesterol and added to mSR-BI cells. Cells take up HDL via the SR-BI scavenger receptor in 2 to more ..
_
   
 Tested Compounds
 Tested Compounds
All(5)
 
 
Active(4)
 
 
Inactive(1)
 
 
 Tested Substances
 Tested Substances
All(5)
 
 
Active(4)
 
 
Inactive(1)
 
 
AID: 588836
Data Source: Broad Institute (2085-12_Inhibitor_SinglePoint_DryPowder_Activity)
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-11-30
Hold-until Date: 2012-11-30
Modify Date: 2012-12-01

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 4
Depositor Specified Assays
AIDNameTypeComment
488952Broad Institute Inihibitors of the HDL Receptor, SR-BI Inhibitors Probe Projectsummary
Description:
Keywords: cholesterol uptake, HDL, SR-BI

Assay Overview:
The goal of this assay is to verify compounds that disrupt the binding of HDL particles to SR-BI scavenger receptor using an alternative means of labeling HDL-cholesterol particles and avoiding any type of fluorescence measurement. To measure this binding event, HDL particles are labeled with the[H3}-cholesterol and added to mSR-BI cells. Cells take up HDL via the SR-BI scavenger receptor in 2 to 3 hours. After significant uptake of the HDL, the radiolabel can be detected by liquid scintillation counting. The level of radioactivity correlates with the amount of HDL uptake. The uptake of lipid particles can be inhibited by the compound BLT-1 or when co-treated with an excess of unlabeled HDL. The mSR-BI cells utilized in the assay are a CHO cell line lacking expression of the LDL receptors and overexpresses the scavenger receptor, SR-BI. Inhibitors of SR-BI and HDL uptake will have a reduction in liquid scinitillation counts.

Expected Outcome: Compounds that inhibit SR-BI mediated uptake will have reduced scinitillation counts
Protocol
Specific cellular uptake of [3H]CE from [3H]CE-HDL were measured for 2 h in the absence (total activity) or presence (non-specific activity) of a 40-fold excess of unlabeled HDL. For experiments, cells were seeded in the wells of 24-well plates (50,000 cells per well) on day 0, and uptake assays were performed on day 2 in Ham's F12K medium plus 0.5% (wtvol) fatty acid free BSA. For uptake assays at 37 degrees C, on the day of the assay, the cells were washed twice with prewarmed (37 degrees C) medium prior to adding radioactive lipo-protein. Specific binding or uptake is the difference between total and nonspecific activities. Data were analyzed using Prism 5 (GraphPad Software). All calculated errors represent standard errors of the mean. In assays BLT-1 (all incubations at 37 degrees C), immediately prior to incubation with radiolabeled lipoproteins, the cells were washed twice with medium, preincubated with BLT-1 for 1 h, and then incubated with radiolabeled lipoproteins as described above, all in medium plus 0.5% (volvol) DMSO in the presence of the indicated concentration of BLT-1. Results were normalized to 100% of control receptor activity in the absence of BLT-1.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1%UPTAKE(1uM) (1μM**)Amount of cholesterol uptake relative to the neutral control, at 1 micromolar concentrationFloat%
2STANDARD_DEVIATIONstandard deviation of tested replicatesFloat%

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

Data Table (Concise)
Classification
PageFrom: