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BioAssay: AID 463206

Luminescence Cell-Based Counter Screen to Identify Inhibitors of Cytokine Induced Apoptosis

The goal of this project is to identify probes that inhibit apoptosis induced by cytokines in the rat insulinoma cell line, INS-1E. The primary assay measures cell viability via ATP levels as a surrogate for apoptosis but does not measure apoptosis directly. A more selective assay is to measure the activity of a direct mediator of apoptosis, such as Caspase 3. INS1-E rat insulinoma cells are more ..
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 Tested Compounds
 Tested Compounds
All(2282)
 
 
Active(167)
 
 
Inactive(2099)
 
 
Inconclusive(16)
 
 
 Tested Substances
 Tested Substances
All(2288)
 
 
Active(167)
 
 
Inactive(2105)
 
 
Inconclusive(16)
 
 
 Related BioAssays
 Related BioAssays
AID: 463206
Data Source: Broad Institute (2061-02_INHIBITORS_DOSE-TITRATION_MLPCN-CHERRYPICK)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-09-07

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 167
Related Experiments
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AIDNameTypeProbeComment
435007Summary of Broad Institute MLPCN Beta Cell Apoptosis Inhibition ProjectSummary1 depositor-specified cross reference: Summary
435005Luminescence Cell-Based Primary HTS to Identify Inhibitors of Beta Cell Apoptosis.Screening same project related to Summary assay
449756Luminescence Cell-Based Dose Retest to Confirm Inhibitors of Beta Cell ApoptosisConfirmatory same project related to Summary assay
463229ATP-based Luminescence in the Absence of Cytokines Measured in Cell-Based System Using Plate Reader - 2061-06_Inhibitor_Dose_CherryPickConfirmatory same project related to Summary assay
488844Detect Cellular ATP-levels in INS-1E Cells Measured in Cell-Based System Using Plate Reader - 2061-01_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
488848Apoptosis Measured in Cell-Based System Using Plate Reader - 2061-02_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
488864ATP-based Luminescence in the Absence of Cytokines Measured in Cell-Based System Using Plate Reader - 2061-06_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
488866Nitrite Measured in Cell-Based System Using Plate Reader - 2061-04_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
488867Apoptosis Measured in Cell-Based System Using Plate Reader - 2061-03_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
488868Nitrite Measured in Cell-Based System Using Plate Reader - 2061-04_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
488870Nitrite Measured in Cell-Based System Using Plate Reader - 2061-04_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatory same project related to Summary assay
488910Detect Cellular ATP-levels in INS-1E Cells Measured in Cell-Based System Using Plate Reader - 2061-01_Inhibitor_SinglePoint_HTS_ActivityScreening same project related to Summary assay
488931Apoptosis Measured in Cell-Based System Using Plate Reader - 2061-03_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
488936Apoptosis Measured in Cell-Based System Using Plate Reader - 2061-02_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
488945Primary Beta Cell Apoptosis assay Measured in Cell-Based System Using Plate Reader - 2061-07_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
488951Primary beta islet insulin ELISA Measured in Cell-Based System Using Plate Reader - 2061-09_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
488959Glucose-induced Insulin secretion ELISA Measured in Cell-Based System Using Plate Reader - 2061-05_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
652090Quantitative proteomics to determine protein binders to ML187Other same project related to Summary assay
652132Effect of ML-187 on cytokines-mediated increase in STAT1 level and activationOther same project related to Summary assay
652171Validation of the affinity reagent probe (ML-187) with a PEG linker: comparison of ML-187-PEG and ML-187 cell-based efficacy Measured in Cell-Based System Using Plate Reader - 2061-11_Inhibitor_Dose_DryPowder_ActivityOther same project related to Summary assay
652206ML-187 activity in a kinase panel for Extended probe characterization for beta-cell apoptosis Measured in Biochemical SystemOther same project related to Summary assay
652234Binding of ML-187 to Usp9x as assessed by surface plasmon resonance Measured in Biochemical SystemOther same project related to Summary assay
652237Effect of siRNA-mediated knock down of binding partners of ML-187 on cytokine-induced apoptosis cell-deathOther same project related to Summary assay
652238Effects of siRNA knock down of Usp9x (a target of ML-187) on cytokine-induced beta-cell death (Cell Titer Glo) Measured in Cell-Based System Using Plate ReaderOther same project related to Summary assay
652240Effects of siRNA knock down of Usp9x (a target of ML-187) on cytokine-induced apoptosis (Caspase activity) Measured in Cell-Based System Using Plate ReaderOther same project related to Summary assay
Description:
Keywords: Type I diabetes, INS-1E cells, Caspase, cytokine, apoptosis

Assay Overview:

The goal of this project is to identify probes that inhibit apoptosis induced by cytokines in the rat insulinoma cell line, INS-1E. The primary assay measures cell viability via ATP levels as a surrogate for apoptosis but does not measure apoptosis directly. A more selective assay is to measure the activity of a direct mediator of apoptosis, such as Caspase 3. INS1-E rat insulinoma cells are treated with 50 ng/mL interferon-gamma, 25 ng/mL Tumor necrosis factor-alpha and 10 ng/mL Interleukin-1-beta for 48 hours in the presence of 100 nL compounds that were leads from the primary screen. A range of concentrations of compound are used from 0.2 uM to 25 uM and IC50 values will be determined. Cells are assayed with Promega's Caspase Glo 3/7, a luciferase-based reagent for measurement of caspase activity.

Expected Outcome:
A compound that prevents the induction of or reduces Caspase-3 activity will decrease the luminescence signal in a dose-dependent manner. These are the type of compounds that will be interesting for further studies. Some compounds will not prevent Caspase activation and will no longer be considered for the project. Compounds should have an IC50 of less than 10 uM.
Protocol
The cytokines induce apoptosis in primary islet beta cells and in the model cell line, INS-1E cells. The primary assay measures cellular viability but not actvity of any components of the apoptosis pathway. This assay measures the levels of Caspase 3 and 7 activity in cells with a luminescence-based assay from Promega called Caspase Glo 3/7. It is known that cytokine treatment leads to a 3 to 4 fold increase in Caspase activity after 48 hours of treatment. INS-1E cells will be treated with cytokines and various concentrations of compounds for 48 hours. The compounds that decrease Caspase activity will be considered for additional studies. In addition, any compound that increases the luminescence signal due to structural properties will be eliminated because these tend to increase the level of luminescence in a dose-dependent manner. The positive control is suberoylanilide hydroxamic acid (SAHA).
Protocol:
Day 0: Collect cells and seed 8,000 cells/well of INS-1E rat beta-cell line in 30 uL using white-walled, bar coded, 384-well plates Corning 8867; incubate at 37 degrees C overnight
Day 1: Add 10 uL medium with cytokine cocktail to each well using the Combi (Cocktail is 250 mL INS1E media, 92.5 uL IFN-gamma, 32.5 uL TNF-alpha and 20 uL IL-1-beta).
Day 3: Add 20 uL Caspase-Glo 3/7 reagent to each well.
Agitate with 'Big Bear' for 15 seconds to maximize cell lysis. Incubate 1 hour at room temperature.
Use Perkin Elmer Envision to read plate luminescence with ultra sensitive settings.
Cell media:
RPMI 1640 (Phenol red free), 5% heat-inactivated fetal calf serum, 1 mM sodium pyruvate, 50 uM 2-mercaptoethanol, 2 mM glutamine, 10 mM HEPES, 100 U/ml penicillin, and 100 ug/ml streptomycin
Cytokines: 10 ng/mL IL-1 beta (R&D Systems, 501-RL-050), 25 ng/mL TNF-alpha (R&D Systems, 410-MT-050), 50 ng/mL IFN-gamma (Sigma, I4777-.1MG)
Comment
Dose Data Analysis
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.
Highest concentration tested: 26 uM
Active concentration limit: 260 uM
In cases where the data points at the highest concentration tested were deemed invalid, the next-highest concentration values were used to determine the active concentration limit.
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 NC wells was set to a normalized activity value of 0.
The median raw signal of the intraplate PC wells was set to a normalized activity value of -100.
Experimental wells were scaled to this range, giving an activity score as percent change in signal relative to the intraplate controls.
Pattern Correction:
No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.
Active Concentration: AC50
Active concentration values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
Active concentration values were calculated up to the active concentration limit.
Curves with constant fits near 0% activity are assumed to have active concentration values outside the active concentration limit.
PUBCHEM_ACTIVITY_OUTCOME:
Activity_Outcome = 1 (inactive) when
a) active concentration > active concentration limit, or
b) compound shows activity but in a direction opposite to the expected outcome
Activity_Outcome = 2 (active) when active concentration <= active concentration 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:
PUBCHEM_ACTIVITY_SCORE is set to 0 when compound is described as 'inactive' in this experiment; that is, curve fit is:
a) constant fit showing near 0% activity at all tested concentrations, or
b) active concentration is calculated to be greater than the active concentration limit, or
c) active but in the opposite direction of the expected outcome in these cases, values describing curve fitting parameters (Sinf, So, Hill Slope, log_AC50, log_AC50_SE) are set to null
PUBCHEM_ACTIVITY_SCORE is set to -10*Log(AC50), where AC50 is in molar, when compound is described as 'active' in this experiment.
Scores relate to active concentrations in this manner:
AC score
1 pM 120
1 nM 90
1 uM 60
1 mM 30
1 M 0
PUBCHEM_ACTIVITY_SCORE is set to 100 when compound is described as 'superactive' in this experiment, that is, curve fit is:
a) constant fit showing full inhibition at all tested concentrations, or
b) active concentration is calculated to be less then lowest tested concentration

Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Cell-based
Assay Cell Type: INS-1
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 EC50 (molar)Float
4Log_AC50_M_Standard_Errorthe standard error for the log of the EC50 valueFloat
5Hill_Slopethe slope at EC50Float
6S0the fitted activity level at zero concentrationFloat%
7SInfthe fitted activity level at infinite concentrationFloat%
8Num_Pointsthe number of data points included in the plotInteger
9Max_Activitythe maximum activity value observedFloat%
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)
Data Table ( Complete ):     View Active Data    View All Data
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