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BioAssay: AID 488951

Primary beta islet insulin ELISA Measured in Cell-Based System Using Plate Reader - 2061-09_Inhibitor_Dose_DryPowder_Activity

This assay measures glucose-induced insulin secretion, the gold standard for beta-cell function. The insulin stimulatory index can be measured by ELISA, after 1-hour incubation with "high glucose" (typically 16.7 mM) in the experimental buffer, in comparison with "low glucose" (typically 1.67 mM). The rat INS-1E cell line is derived from a clone selected for high levels of insulin secretion in more ..
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AID: 488951
Data Source: Broad Institute (2061-09_Inhibitor_Dose_DryPowder_Activity)
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 ):           View Active Data    View All Data
BioActive Compound: 1
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
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
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: insulin, beta cell, ELISA, primary human pancreatic beta islets

Assay Overview:
This assay measures glucose-induced insulin secretion, the gold standard for beta-cell function. The insulin stimulatory index can be measured by ELISA, after 1-hour incubation with "high glucose" (typically 16.7 mM) in the experimental buffer, in comparison with "low glucose" (typically 1.67 mM). The rat INS-1E cell line is derived from a clone selected for high levels of insulin secretion in response to glucose, and thus serves as a good model of beta-cell function. Beta cells treated with cytokines lose their insulin secretory response to glucose. Small molecules that can promote beta-cell survival should restore insulin secretion.
Protocol
Cell and Human Islet Culture. 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 96-well plates containing extracellular matrix secreted by the HTB-9 human bladder carcinoma cell line.
Primary human islets were incubated for 6 days in 100 uL of fresh RPMI containing 1% FBS and the cytokine cocktail, in the presence or absence of compounds. Media was refreshed after 3 days. Cells were washed and incubated for 2 h in KRBH (135 mM NaCl, 3.6 mM KCl, 5 mM NaHCO3, 0.5 mM NaH2PO4, 0.5 mM MgCl2, 1.5 mM CaCl2, 10 mM HEPES, pH 7.4, 0.1% BSA) without glucose. Cells were subsequently incubated with KRBH containing 2 or 15 mM glucose for 1 hour. The supernatant was taken for measurement of released insulin. Insulin was measured with a rat insulin ELISA kit (Alpco).
ALPCO INSULIN ELISA PROTOCOL
Bring all reagents and microplate strips to room temperature prior to use. Gently mix all reagents before use. A standard curve must be performed with each assay and with each microplate if more than one is run at a time. All standards, samples, and the control should be run in duplicate.
1. Ensure that microplates are at room temperature prior to opening foil pouch. Designate enough microplate strips for the standards, desired number of samples, and control. The remaining strips should be stored at 2-8 deg C in the tightly sealed foil pouch containing the desiccant.
2. Pipette 5 ul of each standard, reconstituted control (see Reagent Preparation), or sample into its respective wells.
3. Pipette 75 ul of Working Strength Conjugate (see Reagent Preparation) into each well.
4. Incubate for 2 hours, shaking at 700-900 rpm on a horizontal microplate shaker at room temperature (18-25 deg C).
5. Wash the microplate 6 times with Working Strength Wash Buffer (see Reagent Preparation) with a microplate washer. Alternatively, use a wash bottle to fill the wells, and then discard the liquid, inverting and firmly tapping the microplate on absorbent paper towels between washes. After the final wash with either the microplate washer or wsah bottle, remove any residual Wash Buffer and bubbles from the wells by inverting and firmly tapping the microplate on absorbent paper towels (See Microplate Locking Diagram below).
6. Pipette 100 ul of TMB Substrate into each well.
7. Incubate for 15 minutes at room temperature (18-25 deg C) on a horizontal microplate shaker (700-900 rpm).
8. Pipette 100 ul of Stop Solution into each well. Gently shake the microplate to stop the reaction. Remove bubbles before reading with microplate reader.
9. Place the microplate in a microplate reader capable of reading the absorbance at 450nm with a reference wavelength of 620-650nm. The microplate should be analyzed within 30 minutes following the addition of Stop Solution.
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate. The positive control condition was absence of cytokine.
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:
A standard curve run was run on each assay plate using reference material provided in the ELISA kit. Background-subtracted absorbance values were calculated for each well as (Abs450 - Abs620), and used to extrapolate quantity of insulin secreted in micro International Units (uIU) from the standard curve. No further data normalization methods were applied.
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_0.5uM (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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