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BioAssay: AID 488936

Apoptosis Measured in Cell-Based System Using Plate Reader - 2061-02_Inhibitor_Dose_DryPowder_Activity_Set2

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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AID: 488936
Data Source: Broad Institute (2061-02_Inhibitor_Dose_DryPowder_Activity_Set2)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-10-26
Hold-until Date: 2011-06-13
Modify Date: 2011-06-13

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 3
Related Experiments
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AIDNameTypeProbeComment
435007Summary of Broad Institute MLPCN Beta Cell Apoptosis Inhibition ProjectSummary1 depositor-specified cross reference: Summary assay
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
463206Luminescence Cell-Based Counter Screen to Identify Inhibitors of Cytokine Induced 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
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 activity 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 deg 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
PRESENCE OF CONTROLS: Neutral control wells (NC) 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 (HVC) was determined and the active concentration limit (AC_limit) was set to equal (10)(HVC).
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: 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 >= -70% but <= -30%, or
b) The fit was not valid due to poor fit quality.
PUBCHEM_ACTIVITY_SCORE:
PUBCHEM_ACTIVITY_SCORE = 0 when PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive)
PUBCHEM_ACTIVITY_SCORE = (-10)[Log(AC)], where AC is in molar, when PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
PUBCHEM_ACTIVITY_SCORE = 100 when the curve fit is constant and showing full inhibition at all tested concentrations.
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.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Cell-based
Assay Cell Type: INS-1
From ChEMBL:
Assay Type: Functional
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%
10Max_Activity_Conc_uMThe concentration at which the maximum activity is observedFloatμM
11Activity_at_0.195uM (0.195μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity_at_0.38uM (0.38μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity_at_0.42uM (0.42μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity_at_0.75uM (0.75μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity_at_0.8uM (0.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity_at_1.5uM (1.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
17Activity_at_1.6uM (1.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity_at_3uM (3μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
19Activity_at_6uM (6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
20Activity_at_6.8uM (6.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
21Activity_at_12uM (12μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
22Activity_at_13.5uM (13.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
23Activity_at_23.5uM (23.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
24Activity_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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