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BioAssay: AID 435005

Luminescence Cell-Based Primary HTS to Identify Inhibitors of Beta Cell Apoptosis.

Assay Overview: INS1E rat insulinoma cells are similar to pancreatic beta cells because they secrete insulin in response to glucose stimulation. In the primary assay, INS1E cells are treated with 3 cytokines (Interferon-gamma, Tumor necrosis factor-alpha and Interleukin 1-beta) and compounds for 48 hours. The combination of cytokines leads to apoptotic cell death. The level of cell death is inferred my measuring overall ATP levels with Promega's Cell Titer Glo. ..more
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 Tested Compounds
 Tested Compounds
All(303504)
 
 
Active(2435)
 
 
Inactive(300942)
 
 
Inconclusive(128)
 
 
 Tested Substances
 Tested Substances
All(303588)
 
 
Active(2443)
 
 
Inactive(301017)
 
 
Inconclusive(128)
 
 
 Related BioAssays
 Related BioAssays
AID: 435005
Data Source: Broad Institute (2061-01_INHIBITORS_SINGLE-POINT_MLPCN-HTS)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-06-18

Data Table ( Complete ):           Active    All
BioActive Compounds: 2435
Depositor Specified Assays
AIDNameTypeComment
435007Summary of Broad Institute MLPCN Beta Cell Apoptosis Inhibition Projectsummary
449756Luminescence Cell-Based Dose Retest to Confirm Inhibitors of Beta Cell Apoptosisconfirmatory
Description:
Keywords: cytokine-induced apoptosis, pancreatic beta cells, INS1E insulinoma cells, Type I diabetes

Assay Overview: INS1E rat insulinoma cells are similar to pancreatic beta cells because they secrete insulin in response to glucose stimulation. In the primary assay, INS1E cells are treated with 3 cytokines (Interferon-gamma, Tumor necrosis factor-alpha and Interleukin 1-beta) and compounds for 48 hours. The combination of cytokines leads to apoptotic cell death. The level of cell death is inferred my measuring overall ATP levels with Promega's Cell Titer Glo.

Expected Outcome:
Cytokine induced apoptosis leads to cell death and thus, reduced ATP levels. Candidate compounds will prevent cell death and keep ATP levels elevated. ATP is measured with Cell Titer go (Promega) and so increased values are expected. A compound is scored as a hit when both replicates score as 74% activity or higher and has a minimum Z score of 3.
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.

Cell carrying media:
RPMI 1640, 10% 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

Plating 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), 25 ng/mL TNF-alpha (R&D Systems, 410-MT), 50 ng/mL IFN-gamma (R&D 485-MI)
Comment
HTS Data Analysis

Negative control wells (NC) and positive control wells (PC) were included on every plate.
Active compounds result in increased readout signal.

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 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.

The plate pattern correction algorithm 'Runwise Pattern (Multiplicative)' in Genedata (v7.0.3) was applied to the normalized plate data.

The final PUBCHEM_ACTIVITY_SCORE was set as equal to the mean of the replicate percent activities.

The PUBCHEM_ACTIVITY_OUTCOME class was assigned as described below, based on an activity threshold of 75%:

Activity_Outcome = 1 (inactive)
Less than half of the replicates fell outside the threshold.

Activity_Outcome = 2 (active)
All of the replicates fell outside the threshold,
OR
At least half of the replicates fell outside the threshold AND the PUBCHEM_ACTIVITY_SCORE fell outside the threshold.

Activity_Outcome = 3 (inconclusive)
At least half of the replicates fell outside the threshold AND the PUBCHEM_ACTIVITY_SCORE did not fall outside the threshold.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1REPRODUCIBILITY_COSINE_TRANSFORMA measure of how well the activity reproduced across the two samples. Computed as the absolute value of the cosine between the "replicate vector" (ScoreA, ScoreB ---as well as ScoreC and/or ScoreD where applicable) and the vector (1, 1) representing perfect reproducibility. NULL will appear in this column if a sample was not run in duplicate or if the data produced by one of the replicates was InvalidFloat
2BROAD_SCREENING_RUNIDSThis is a comma separated list of unique IDs given to each screening run at the Broad Institute.String
3REPLICATE_A_ACTIVITY_SCOREThe calculated activity score for the indicated sample, a NULL here denotes data was not used or was not producedFloat%
4REPLICATE_B_ACTIVITY_SCOREThe calculated activity score for the indicated sample, a NULL here denotes data was not used or was not producedFloat%
5REPLICATE_C_ACTIVITY_SCOREThe calculated activity score for the indicated sample, a NULL here denotes data was not used or was not producedFloat%
6REPLICATE_D_ACTIVITY_SCOREThe calculated activity score for the indicated sample, a NULL here denotes data was not used or was not producedFloat%
7DATE_REPORTEDThe date the data was internally reportedString
Additional Information
Grant Number: DP2 DK083048

Data Table (Concise)
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