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BioAssay: AID 463229

ATP-based Luminescence in the Absence of Cytokines Measured in Cell-Based System Using Plate Reader - 2061-06_Inhibitor_Dose_CherryPick

This is a counter-screen to test compounds for ATP-based luminescence in the absence of cytokines.Cells will be treated with compounds at various doses for 48 hours. The cells will not be treated with cytokines. While perhaps interesting in another context, compounds that elevate ATP levels in INS-1E cells under basal conditions are not suitable as probes in this assay. Further, compounds that suppress cytokine-induced beta-cell apoptosis but are toxic in their own right are similarly unattractive. ..more
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 Tested Compounds
 Tested Compounds
All(2282)
 
 
Active(2059)
 
 
Inactive(223)
 
 
 Tested Substances
 Tested Substances
All(2288)
 
 
Active(2065)
 
 
Inactive(223)
 
 
 Related BioAssays
 Related BioAssays
AID: 463229
Data Source: Broad Institute (2061-06_Inhibitor_Dose_CherryPick)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-09-17

Data Table ( Complete ):           Active    All
BioActive Compounds: 2059
Depositor Specified Assays
AIDNameTypeComment
435007Summary of Broad Institute MLPCN Beta Cell Apoptosis Inhibition ProjectsummaryProject Summary
Description:
Keywords:
INS1E, cytotoxicity, Cell titer glo, cellular ATP

Assay Overview:
This is a counter-screen to test compounds for ATP-based luminescence in the absence of cytokines.Cells will be treated with compounds at various doses for 48 hours. The cells will not be treated with cytokines. While perhaps interesting in another context, compounds that elevate ATP levels in INS-1E cells under basal conditions are not suitable as probes in this assay. Further, compounds that suppress cytokine-induced beta-cell apoptosis but are toxic in their own right are similarly unattractive.
Protocol
Protocol:
Day 0: Collect cells and generate single cell suspension by trypsinization and passing through sterile 40 micron cell strainer (Falcon). Seed 8,000 cells/well of INS-1E rat beta-cell line in 30 uL media using white, opaque, bar coded, 384-well Corning 8867 plates; incubate at 37 degrees C overnight
Day 1: Add 10 uL medium with cytokine cocktail to each well using the Combi. Pin transfer compounds to plates right after the addition of cyokines with 100 nL head and transfer 100 nL compound. Positive control added by double pinning plates.
Day 3: Add 20 uL Cell titer-Glo reagent to plates.
Agitate gently for 15 seconds to maximize cell lysis. Incubate 8 minutes.
Use Envision to read plate luminescence with standard luminescence parameters.
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.

EXPECTED OUTCOME: Active compounds result in increasing readout signal.

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

NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Stimulators Minus Neutral Controls' 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: No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.

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.

PUBCHEM_ACTIVITY_OUTCOME:
Activity_Outcome = 1 (inactive) when:
a) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
b) AC > AC_limit, or
c) 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

Activity_Outcome = 2 (active) when:
AC <= AC_limit

Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= 30% but <= 70%, or
b) The fit was not valid due to poor fit quality.

PUBCHEM_ACTIVITY_SCORE:
Activity_Score = 0 when Activity_Outcome = 1 (inactive) or 3 (inconclusive)

Activity_Score = (-10)[Log(AC)], where AC is in molar, when Activity_Outcome = 2 (active)
Scores relate to AC in this manner:
AC score
1 pM 120
1 nM 90
1 uM 60
1 mM 30
1 M 0

Activity_Score = 100 when the curve fit is constant and showing full inhibition at all tested concentrations.
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
3Log_AC50_MThe log of the molar AC50Float
4Log_AC50_M_Standard_ErrorThe standard error for the log of the molar AC50 valueFloat
5Hill_SlopeThe slope at AC50Float
6S0The fitted activity value at zero concentrationFloat%
7SinfThe fitted activity value at infinite concentrationFloat%
8Num_PointsThe number of data points used to generate the plotInteger
9Max_ActivityThe maximum activity value observed, based on mean of replicates per concentrationFloat%
10Activity_at_0.195uM (0.195μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
11Activity_at_0.38uM (0.38μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity_at_0.8uM (0.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity_at_1.6uM (1.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity_at_3uM (3μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity_at_6uM (6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity_at_12uM (12μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
17Activity_at_26uM (26μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: DP2 DK083048

Data Table (Concise)
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