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BioAssay: AID 488867

Apoptosis Measured in Cell-Based System Using Plate Reader - 2061-03_Inhibitor_Dose_DryPowder_Activity_Set2

In apoptosis, electron transport is disrupted, cytochrome c is released, and the mitochondroal membrane and the electrochemical potential across the mitochondrial membrane becomes reduced. In a normal, mitochondria, JC-1 is a cationic dye which exhibits a membrane potential-dependent accumulation in the mitochondrion. The dye exists as a monomer at low concentrations and yields green more ..
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 Tested Compounds
 Tested Compounds
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Active(15)
 
 
Inactive(72)
 
 
 Tested Substances
 Tested Substances
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Active(15)
 
 
Inactive(73)
 
 
 Related BioAssays
 Related BioAssays
AID: 488867
Data Source: Broad Institute (2061-03_Inhibitor_Dose_DryPowder_Activity_Set2)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-10-22
Hold-until Date: 2011-06-13
Modify Date: 2011-06-13

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 15
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
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: Mitochondra, INS-1E, type I diabetes, JC-1

Assay Overview:

In apoptosis, electron transport is disrupted, cytochrome c is released, and the mitochondroal membrane and the electrochemical potential across the mitochondrial membrane becomes reduced. In a normal, mitochondria, JC-1 is a cationic dye which exhibits a membrane potential-dependent accumulation in the mitochondrion. The dye exists as a monomer at low concentrations and yields green fluorescence, similar to fluorescein. At higher concentrations, the dye forms J-aggregates that exhibit a broad excitation spectrum and an emission maximum at ~590 nm (i.e. red fluorescence). In apoptotic cells, JC-1 remains in the cytoplasm as the green monomer. Mitochondrial depolarization is indicated by a decrease in the red to green intensity ratio. The color shift is attributed to the concentration dependant accumulation of red fluorescent J-aggregates. In this assay, INS-1E insulinoma cells will be treated with 10 ng/mL IL-1 beta, 50 ng/mL IFN-gamma, 25 ng/mL TNF-alpha for 48 hours in the presence of several doses of compounds.
Protocol
Protocol:
1. Seed cells into black 384-well optical bottom plates at 8000 cells per well using 50mL/well with Combi.
2. Change media to 50uL/well prior to compound addition (usually on day 2). After overnight incubation, medium was removed and 50uL RPMI containing 1% FBS and a combination of cytokines (10ng/mL IL-1b, 50ng/mL IFN-g, 25ng/mL TNF-a) was added to every well. Using libraries of compounds dissolved in DMSO and a CyBi-Well pin-transfer robot (CyBio Corp.), 0.1 uL of each compound was added to the wells.
3. After incubation for 48 hr, medium was removed. Dilute 1mM JC-1 stock in phenol-red free media to a final concentration of 3.25mM. Aspirate plates, and add 20mL/well.
4. Incubate plates at 37 deg C for 2 hours.
5. Wash cells three times with 50ul/well of 1X PBS.
6. Read fluorescence on the Envision plate reader using the rhodamine channel (ex/em 530/580) followed by the fluorescein channel (ex/em 485nm/530nm). Each time a new assay is read, run an optimization cycle on the instrument to ensure the appropriate reading levels for the assay.
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC) 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 (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 >= 30% but <= 70%, 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 ChEMBL:
Assay Format: Cell-based
Assay Type: Functional
Assay Cell Type: INS1
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)
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