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BioAssay: AID 488945

Primary Beta Cell Apoptosis assay Measured in Cell-Based System Using Plate Reader - 2061-07_Inhibitor_Dose_DryPowder_Activity_Set2

The best indication of physiological relevance in cell culture is to recapitulate our results in human beta cells. Therefore, we will test the effects of promising screening positives in primary human pancreatic islets. We have access to human islets through the Juvenile Diabetes Research Foundation, and have confirmed that dissociated human islet cells are sensitive to cytokine treatment. more ..
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 Tested Compounds
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Active(1)
 
 
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Active(1)
 
 
 Related BioAssays
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AID: 488945
Data Source: Broad Institute (2061-07_Inhibitor_Dose_DryPowder_Activity_Set2)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2010-10-27
Hold-until Date: 2011-06-13
Modify Date: 2011-06-13

Data Table ( Complete ):           Active    All
BioActive Compound: 1
Depositor Specified Assays
AIDNameTypeComment
435007Summary of Broad Institute MLPCN Beta Cell Apoptosis Inhibition ProjectsummarySummary assay
Description:
Keywords: ELISA, insulin, apoptosis, primary human beta cells

Assay Overview:
The best indication of physiological relevance in cell culture is to recapitulate our results in human beta cells. Therefore, we will test the effects of promising screening positives in primary human pancreatic islets. We have access to human islets through the Juvenile Diabetes Research Foundation, and have confirmed that dissociated human islet cells are sensitive to cytokine treatment. Primary human pancreatic islets containing beta cells are cultured in the presence of cytokines (IFN, TNF and IL1Beta) for 3 and 6 days. They are exposed to high glucose to stimulate insulin production. Supernatants are collected for the insulin ELISA while cells are tested for apoptotic activity with the Caspase-glo 3/7 assay (Promega). Compounds that protect beta cells as measured by restoration of insuliation secretion will be suitable for probe declaration.
Protocol
Protocol:

Human islets were obtained through the Islet Cell Resource Consortium (http://icr.coh.org/) and through the National Disease Research Interchange (http://www.ndriresource.org/). The purity and viability of human islets are reported to be 70-93% and 70-98%, respectively, and the average age of cadaveric donors was 40.7 +/- 9.0 y (range 32-57 y; n = 6). Islets were washed with PBS and incubated in CMRL medium supplemented with 10% FBS, 2 mM glutamine, 100 U/mL penicillin, and 100 ug/mL streptomycin. Islets were gently dissociated into a cell suspension by incubating in Accutase (37 deg C, 10 min), and seeded in 384-well plates containing extracellular matrix secreted by the HTB-9 human bladder carcinoma cell line.

Primary human islets were incubated for 6 days in 50 uL of fresh RPMI containing 1% FBS and the cytokine cocktail, in the presence or absence of compounds. Media was changed after 3 days. After 6 days, an equal volume of Caspase-glo 3/7 (Promega) was added per well and incubated at room temperature for 2 hours. After 2 hours, luminescence was measured with the Perkin-Elmer Envison plate reader.
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.
NC wells contained DMSO only (no compound).
PC wells contained DMSO only and no cytokine.

NORMALIZATION:
Plate well values (W) were normalized according to this formula: ((W - NC_mean) / (PC_mean - NC_mean))

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).

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.
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_0.5uM (0.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity_at_1uM (1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity_at_2uM (2μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity_at_5uM (5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity_at_10uM (10μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity_at_20uM (20μM**)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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