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BioAssay: AID 488896

HTS 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_Activity

HDL particles are labeled with DiI and added to mSR-B1 cells at 10 ug/mL in black, 384 well plates. Cells normally take up HDL via the SR-B1 scavenger receptor in 2 to 3 hours. After significant uptake of the DiI-HDL, cells become fluorescent. The level of fluorescence correlates with the amount of HDL uptake and can be measured with the Perkin Elmer Envision plate reader. The uptake can be more ..
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 Tested Compounds
 Tested Compounds
All(319195)
 
 
Active(3064)
 
 
Inactive(315524)
 
 
Inconclusive(613)
 
 
 Tested Substances
 Tested Substances
All(319533)
 
 
Active(3066)
 
 
Inactive(315854)
 
 
Inconclusive(613)
 
 
AID: 488896
Data Source: Broad Institute (2085-01_Inhibitor_SinglePoint_HTS_Activity)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-10-25
Modify Date: 2010-11-16

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 3064
Depositor Specified Assays
AIDNameTypeComment
488952Broad Institute Inihibitors of the HDL Receptor, SR-BI Inhibitors Probe Projectsummary
Description:
Keywords: HDL, Scavenger receptor, SR-BI, cholesterol

Assay Overview:
HDL particles are labeled with DiI and added to mSR-B1 cells at 10 ug/mL in black, 384 well plates. Cells normally take up HDL via the SR-B1 scavenger receptor in 2 to 3 hours. After significant uptake of the DiI-HDL, cells become fluorescent. The level of fluorescence correlates with the amount of HDL uptake and can be measured with the Perkin Elmer Envision plate reader. The uptake can be inhibited with the compound BLT-1 or the fluorescence can be reduced when co-treated with an excess of unlabeled HDL. mSR-BI cells are a CHO cell line that lacks expression of the LDL receptors and overexpresses the scavenger receptor, SR-BI. mSR-BI cells are exposed to compound for 3 hours. DiI labeled HDL is added to the well at a final of 10 ug/mL. Inhibitors of SR-B1 and HDL uptake will have a reduction in fluorescence.
Protocol
Protocol:

Day 0:
Plate 10,000 cells ldlA[mSR-BI] 30ul/ well in Ham's F12/10% FCS/PSG

Day 1:
1) Remove media with aspirator.
2) Add 30ul Ham's F12/0.5% BSA/25Mm HEPES pH 7.4 + 10 ug/mL DiI-HDL with Combi
3) Pin transfer 100 nl compounds and positive control (sentinel plate).
4) Incubate 3 hours @ 37 degrees C
5) Remove media with aspirator.
6) Analyze DiI-HDL uptake with Envision Bodipy TMR mirror #405, Excitation filter is Photometric 550 (#312) and emission filter is Cy3 595 (#229) with bottom read
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.

EXPECTED OUTCOME: Active compounds result in decreasing readout signal.

NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.

PATTERN CORRECTION: The plate pattern correction algorithm 'Runwise Pattern (Multiplicative)' in Genedata (v7.0.3) was applied to the normalized plate data.

PLATE MASKING: Plate with a Z' factor < 0.3 were excluded from further analysis

PUBCHEM_ACTIVITY_SCORE:
This was set as equal to the mean of the normalized and corrected sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at 70.

PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 75.

PUBCHEM_ACTIVITY_OUTCOME:

Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T

Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T

Samples passing AT only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T

Samples passing PAR_T only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1REPRODUCIBILITY_COSINE_TRANSFORMA measure of how well the activity reproduced across the two samples. Computed as the absolute value of the cosine between the 'replicate vector' (ScoreA, ScoreB ---as well as ScoreC and/or ScoreD where applicable) and the vector (1, 1) representing perfect reproducibility. NULL will appear in this column if a sample was not run in duplicate or if the data produced by one of the replicates was InvalidFloat
2REPLICATE_A_ACTIVITY_SCORE (12.5μM**)The calculated percent activity for the indicated sampleFloat%
3REPLICATE_B_ACTIVITY_SCORE (12.5μM**)The calculated percent activity for the indicated sampleFloat%
4REPLICATE_C_ACTIVITY_SCORE (12.5μM**)The calculated percent activity for the indicated sampleFloat%
5REPLICATE_D_ACTIVITY_SCORE (12.5μM**)The calculated percent activity for the indicated sampleFloat%
6REPLICATE_E_ACTIVITY_SCORE (12.5μM**)The calculated percent activity for the indicated sampleFloat%
7REPLICATE_F_ACTIVITY_SCORE (12.5μM**)The calculated percent activity for the indicated sampleFloat%
8REPLICATE_G_ACTIVITY_SCORE (12.5μM**)The calculated percent activity for the indicated sampleFloat%
9REPLICATE_H_ACTIVITY_SCORE (12.5μM**)The calculated percent activity for the indicated sampleFloat%

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

Data Table (Concise)
Classification
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