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BioAssay: AID 540246

HDL Counter Assay: 3/24 Cytotox Measured in Cell-Based System Using Plate Reader - 2085-02_Inhibitor_Dose_DryPowder_Activity

The HDL uptake primary assay is looking for a decrease in fluorescence. It is possible that a compound will cause cells to decrease HDL uptake due to non-specific consequences of cellular toxicity. Therefore, we would like to make sure that lead compounds are not toxic in the 3 hour time period used in the primary assay. Cells are treated with compounds for 3 or 24 hours and then cell viability more ..
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 Tested Compounds
 Tested Compounds
All(65)
 
 
Active(9)
 
 
Inactive(56)
 
 
 Tested Substances
 Tested Substances
All(65)
 
 
Active(9)
 
 
Inactive(56)
 
 
AID: 540246
Data Source: Broad Institute (2085-02_Inhibitor_Dose_DryPowder_Activity)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-07-13

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 9
Depositor Specified Assays
AIDNameTypeComment
488952Broad Institute Inihibitors of the HDL Receptor, SR-BI Inhibitors Probe ProjectsummarySummary assay
Description:
Keywords: cytotoxicity, Cell titer glo, ldlA[SRB1] cells


Assay Overview:

The HDL uptake primary assay is looking for a decrease in fluorescence. It is possible that a compound will cause cells to decrease HDL uptake due to non-specific consequences of cellular toxicity. Therefore, we would like to make sure that lead compounds are not toxic in the 3 hour time period used in the primary assay. Cells are treated with compounds for 3 or 24 hours and then cell viability is measured using the Cell Titer Glo Assay which is a luciferase-based reagent that measures cellular ATP levels. Compounds will be tested at 8 different doses to determine IC50 values. Compounds that are toxic after 3 hours at IC50>30 uM will be excluded from additional studies. Compounds that are active in the primary assay but toxic below 30 uM at 24 hours will still be pursued.

Expected Outcome:

Cells that are cytotoxic will decrease cellular ATP levels resulting in a decrease in luminescence.
Protocol

Protocol:

Day 0:
Plate 4,000 cells ldlA[mSR-BI] 30microl/ well in Ham's F12/10% FCS/PSG

Day 1:
1)Pin transfer 100 nl compounds and positive control (sentinel plate).
2)Incubate 3 hours @ 37 degrees C
3)Add 20 uL Promega Cell titer glo per well
4)Shake plate for 15 seconds
5)Incubate 10 minutes
6)Read luminescence with Perkin-Elmer Envision plate reader
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC; n=32) and positive control wells (PC; n=32) were included on every plate.

EXPECTED OUTCOME: Active compounds result in decreasing readout signal.

ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal (10)(Max_Concentration).

NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral controls (NC) is set to a normalized activity value of 0.
A normalized activity value of 100 is defined as (2)(NC).
A normalized activity value of -50 is defined as (0.5)(NC).
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.

MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): AC50
AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.
pAC was set to equal -1*log10(AC)

PUBCHEM_ACTIVITY_OUTCOME:
Activity_Outcome = 1 (inactive) when:
a) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null
b) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
c) AC > AC_limit
Activity_Outcome = 2 (active) when:
AC <= AC_limit
Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= -70% but <= -30%, or
b) The fit was deemed not valid due to poor fit quality.

PUBCHEM_ACTIVITY_SCORE:
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
When the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.
When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.

Note:
The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.
All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1AC50_Qualifier'>','=', or '<'String
2AC50_uM*The concentration at which activity reaches 50% of the maximumFloatμM
3pAC50_MEqual to -1*log10(AC50)String
4Hill_SlopeThe slope at AC50Float
5S0_(%)The fitted activity value at zero concentrationFloat%
6Sinf_(%)The fitted activity value at infinite concentrationFloat%
7Num_PointsThe number of data points used to generate the plotInteger
8Max_Activity_(%)The maximum activity value observed, based on mean of replicates per concentrationFloat%
9Max_Activity_Conc_uMThe concentration at which the maximum activity is observedFloatμM
10Max_Concentration_uMMaximum valid test concentrationFloatμM
11Activity_at_0.0135uM_(%) (0.0135μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity_at_0.015uM_(%) (0.015μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity_at_0.016uM_(%) (0.016μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity_at_0.03uM_(%) (0.03μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity_at_0.042uM_(%) (0.042μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity_at_0.046uM_(%) (0.046μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
17Activity_at_0.05uM_(%) (0.05μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity_at_0.09uM_(%) (0.09μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
19Activity_at_0.12uM_(%) (0.12μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
20Activity_at_0.135uM_(%) (0.135μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
21Activity_at_0.15uM_(%) (0.15μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
22Activity_at_0.285uM_(%) (0.285μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
23Activity_at_0.38uM_(%) (0.38μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
24Activity_at_0.42uM_(%) (0.42μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
25Activity_at_0.8uM_(%) (0.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
26Activity_at_1.1uM_(%) (1.1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
27Activity_at_1.2uM_(%) (1.2μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
28Activity_at_1.35uM_(%) (1.35μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
29Activity_at_2.35uM_(%) (2.35μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
30Activity_at_3.5uM_(%) (3.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
31Activity_at_3.8uM_(%) (3.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
32Activity_at_7.5uM_(%) (7.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
33Activity_at_10uM_(%) (10μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
34Activity_at_11uM_(%) (11μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
35Activity_at_12uM_(%) (12μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
36Activity_at_21uM_(%) (21μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
37Activity_at_30uM_(%) (30μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
38Activity_at_35uM_(%) (35μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
39Activity_at_68uM_(%) (68μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%

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

Data Table (Concise)
Classification
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