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BioAssay: AID 493234

Hypoxia inducible factor pathway Zinc Measured in Cell-Based System Using Plate Reader - 2030-05_Activator_Dose_DryPowder_Activity_Set2

Keywords: Hypoxia, Hypoxia Inducible Factor (HIF), Hypoxia Responsive Element (HRE), luciferase, transcriptional activation, tissue regeneration, ischemia ..more
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 Tested Compounds
 Tested Compounds
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Active(39)
 
 
Inactive(50)
 
 
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 Tested Substances
All(90)
 
 
Active(40)
 
 
Inactive(50)
 
 
 Related BioAssays
 Related BioAssays
AID: 493234
Data Source: Broad Institute (2030-05_Activator_Dose_DryPowder_Activity_Set2)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-02-16
Hold-until Date: 2012-02-15
Modify Date: 2012-02-15

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 39
Related Experiments
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AIDNameTypeComment
1971Broad Institute MLPCN Hypoxia Inducible Factor Activation ProjectSummarydepositor-specified cross reference: Summary assay
1910Luminescence Cell-Based Primary HTS to Identify Transcriptional Activators of Hypoxia-Inducible Factor PathwayScreeningsame project related to Summary assay
2089Luminescence Cell-Based Confirmation at Dose to Identify Transcriptional Activators of Hypoxia-Inducible Factor PathwayConfirmatorysame project related to Summary assay
2486Luminescent Cell-Based Dose Titration Retest Counterscreen to Identify Proteasome InhibitorsConfirmatorysame project related to Summary assay
434951Fluorescent Cell-Based Secondary Screen to Identify Activators of the Hypoxia Factor PathwayConfirmatorysame project related to Summary assay
488804Proteasome Measured in Cell-Based System Using Plate Reader - 2030-02_Activator_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
488805Hypoxia inducible factor pathway Measured in Cell-Based System Using Plate Reader - 2030-01_Activator_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
488820Hypoxia Inducible pathway Measured in Cell-Based System Using Imaging - 2030-03_Activator_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
488843HIF pathway Measured in Cell-Based System Using RT-PCR - 2030-04_Activator_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
493180Hypoxia inducible factor pathway Measured in Cell-Based System Using Plate Reader - 2030-01_Activator_Dose_DryPowder_Activity_Set3Confirmatorysame project related to Summary assay
493193Hypoxia inducible factor pathway Measured in Cell-Based System Using Plate Reader - 2030-01_Activator_Dose_DryPowder_Activity_Set4Confirmatorysame project related to Summary assay
493195Hypoxia inducible factor pathway Measured in Cell-Based System Using Plate Reader - 2030-01_Activator_Dose_DryPowder_Activity_Set6Confirmatorysame project related to Summary assay
493213Counterscreen Measured in Cell-Based System Using Plate Reader - 2030-02_Activator_Dose_DryPowder_Activity_Set3Confirmatorysame project related to Summary assay
493225Hypoxia inducible pathway Measured in Cell-Based System Using Imaging - 2030-03_Activator_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
493227Hypoxia inducible factor pathway Measured in Cell-Based System Using Plate Reader - 2030-01_Activator_Dose_DryPowder_Activity_Set8Confirmatorysame project related to Summary assay
493228Hypoxia inducible factor pathway Iron Measured in Cell-Based System Using Plate Reader - 2030-06_Activator_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
493230HIF pathway Measured in Cell-Based System Using RT-PCR - 2030-04_Activator_Dose_DryPowder_Activity_Set3Confirmatorysame project related to Summary assay
493235Hypoxia inducible factor pathway Measured in Cell-Based System Using Plate Reader - 2030-01_Activator_Dose_DryPowder_Activity_Set7Confirmatorysame project related to Summary assay
493236Counterscreen Measured in Cell-Based System Using Plate Reader - 2030-02_Activator_Dose_DryPowder_Activity_Set4Confirmatorysame project related to Summary assay
493237Hypoxia inducible factor pathway Iron Measured in Cell-Based System Using Plate Reader - 2030-06_Activator_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
493238HIF pathway Measured in Cell-Based System Using RT-PCR - 2030-04_Activator_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
493239Hypoxia inducible pathway Measured in Cell-Based System Using Imaging - 2030-03_Activator_Dose_DryPowder_Activity_Set3Confirmatorysame project related to Summary assay
493249Hypoxia inducible factor pathway Measured in Cell-Based System Using Plate Reader - 2030-01_Activator_Dose_DryPowder_Toxicity_Set7Confirmatorysame project related to Summary assay
504323Hypoxia inducible factor pathway Zinc Measured in Cell-Based System Using Plate Reader - 2030-05_Activator_Dose_DryPowder_ToxicityConfirmatorysame project related to Summary assay
504324Hypoxia inducible factor pathway Zinc Measured in Cell-Based System Using Plate Reader - 2030-05_Activator_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
504331Hypoxia inducible factor pathway Iron Measured in Cell-Based System Using Plate Reader - 2030-06_Activator_Dose_DryPowder_ToxicityConfirmatorysame project related to Summary assay
504588Hypoxia inducible factor pathway Measured in Cell-Based System Using Plate Reader - 2030-01_Activator_Dose_DryPowder_Activity_Set10Confirmatorysame project related to Summary assay
504597Hypoxia inducible factor pathway Measured in Cell-Based System Using Plate Reader - 2030-01_Activator_Dose_DryPowder_Activity_Set9Confirmatorysame project related to Summary assay
Description:
Keywords: Hypoxia, Hypoxia Inducible Factor (HIF), Hypoxia Responsive Element (HRE), luciferase, transcriptional activation, tissue regeneration, ischemia

Assay Overview: Luciferase assay (Steady-Glo, Promega).
Primary screen using human osteosarcoma U2OS cells stably over-expressing a plasmid containing 3 copies of the Hypoxia Responsive Element linked to luciferase gene (U2OS HRE-luciferase cells) to identify small molecules inducing an increased luciferase activity in these cells. The small molecules inducing expression of luciferase under HRE promoter will be measured by the conversion of luciferin to oxyluciferin and light (luminescence) mediated by the luciferase.

Expected Outcome: Most known HIF inducers are metal chelators. In this experiment, we are testing the ability of these HIF inducer compounds to chelate metal(s) (zinc or iron). For compounds that are metal chelators, the dose response curve generated by these compounds should shift to the right in the medium exposed to a metal (zinc or iron) versus the medium without metal. Compounds where their absolute values (absciss values, ABSCNNs) are crossing the linear portion of the majority of the compound dose response curves (EC50) is shifting by 10 fold or more to the right and show a reduction of the maximum activity (Sinf) of at least 2 fold will be considered iron and/or zinc chelators. Compounds showing no toxicity (no IC50) in a cell viability test (Celltiter-Glo, Promega) but shifting to the right the ABSCNN value (10 fold) in the gene reporter luciferase assay will be considered as iron and/or zinc chelators.
Protocol
Protocol:
U2OS HRE-luciferase assay:
The U2OS-HRE-luc cell line was originally obtained from Dr. Margaret Ashcroft's lab and kindly provided by Dr. Shawn Gilbert.
The U2OS-HRE-luc cell line is propagated in DMEM media (Invitrogen, SKU# 11995) supplemented with 10% heat inactivated fetal bovine serum (Invitrogen, Cat# 16140089), 1% penicillin/streptomycin/glutamine (Invitrogen, 10378-016) and 0.1 mg/ml Hygromycin B (Invitrogen, 10687-01) at 37degrees C in CO2 incubators (Thermo Scientific) with 5% CO2, 21% O2, and 95% humidity. For High-Throughput Screening assays, cells are grown in T175 flask (BD Falcon, Ref# 353112) or Hyperflasks (Corning, Cat# 10010), harvested at more than 80% confluence using 7 or 75 ml Trypsin-EDTA 0.25% (Cellgro, Cat# 25-053-CI) for 5 minutes and then the trypsin is inactivated with 7 or 75 ml of complete medium respectively. Cells should not be trypsinized more than 10 minutes because massive cell death will start to occur. Cells are centrifuged at 1000rpm/5min and resuspended in fresh complete DMEM media with phenol red (Invitrogen, SKU# 11995) as mentioned above (for normal cell propagation) or DMEM medium without phenol red (Cambrex; Cat# 12-917F or Invitrogen cat# 31053) with 10% Heat inactivated fetal bovine serum (Invitrogen, Cat# 16140089), 1% penicillin/streptomycin/glutamine (Invitrogen, 10378-016), sodium pyruvate (only used with DMEM without phenol red from Invitrogen (Cat# 11306-070)) and without hygromycin B (for compound screening). Cell number is counted using a Nexelcom Bioscience cell counter (Cellometer Auto M10) and viability is measured by mixing one volume of cells with one same volume of Trypan Blue solution (0.4%)(dilution 1/2). Only cultures of >94% viability are utilized for HTS.
Compound Screening is carried out on the Broad Institute/Chemical Biology Platform Walkup unit:
Day 1 (Cell plating):
1. U2OS-HRE-luc cells are harvested and resuspended in DMEM without phenol red (Cambrex, 12-917F) with 10% Heat inactivated fetal bovine serum (Invitrogen, 16140089), 1% penicillin/streptomycin/glutamine (Invitrogen,10378-016) without hygromycin B. U2OS HRE-luciferase cells (from an initial cell suspension of 120,000 cells/ml) are dispensed using a MultiDrop Combi/Standard tube dispensing cassette (Thermo Scientific) in white bottom 384 well assay plates (Corning, Cat.No. 8867BC) at a final density of 6,000 cells per well in final volume of 50 uL. The cells are kept in suspension using a magnetic bar and a stirrer during the dispensing.
2. The assay plate (cell plate) are placed in Liconic Instruments cassettes (22 plates/cassette) and incubated for 24 hours at 37 degrees C in the Liconic CO2 incubator (Liconic Instruments) calibrated at 5% CO2, 21% O2, and 95% humidity.
Day 2 (Compound pinning into assay plate):
3. Before pinning the compounds, 10 ul of 2.5mM Zinc sulfate (Fluka, 96495) was added to each well to a final concentration of 50 uM. The dose response compound plate (8 concentrations, 3 fold dilution, starting concentration 7.5 mM) with the positive control (Desferrioxamine, 80 mM)(32 wells spread across the plate) are pinned twice using 384 well pin tool (100 nl) on pin table (Walkup Cybi Well) and transferred to assay plate. Pins are washed with methanol and DMSO between each pinning.
4. The assay plates treated with compounds are moving back to Liconic CO2 incubator to be incubated for an additional 24 hours.
Day 3 (Reading luminescence from assay plates with Envision):
5. Each assay plate is pulled out of the incubator and cooled down at room temperature for 30 minutes. 30 uL/well (384 well) of Steady-Glo luciferase reagent 0.5X (diluted in H2O) (Promega, E2550) is dispensed using the the MultiDrop Combi dispensing cassette from Thermo Scientific. For the toxicity (cell viability) readout, 30 uL/well (384 well) of Celltiter-Glo luciferase reagent 0.5X (diluted in H2O) (Promega, G7573) is dispensed using the the MultiDrop Combi dispensing cassette from Thermo Scientific. The assay plate returned to room temperature for 30 minutes to allow a complete cellular lysis.
6. Luminescence is measured (0.2 second/well) using the ultra sensitive luminescence detector (384-well aperture, 0.5 mm height) in each well using the Envision plate reader (Perkin Elmer)(Corning plate setting).
7. HRE activation is calculated based on the following equation using mean luminescence (RLU) values:
Fold Activation = (Treatment - Background)/(No treatment - Background)
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC; n=32) and positive control wells (PC; n=32) 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 (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal Max_Concentration.
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): absACnn, the concentration at which the curve crosses threshold 500.0
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.
pAC was set to equal -1*log10(AC)
PUBCHEM_ACTIVITY_OUTCOME:
Activity_Outcome = 1 (inactive) when:
a) 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
b) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
c) AC > AC_limit
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 deemed not valid due to poor fit quality.
PUBCHEM_ACTIVITY_SCORE:
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
When the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.
When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.
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: U-2 OS
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1AbsAC500_Qualifier<, =, or >String
2AbsAC500_uM*The concentration at which the fitted curve passes activity threshold 500.FloatμM
3pAbsAC500_MEqual to -1*log10(AbsAC500).Float
4Hill_SlopeThe slope at AC50Float
5S0_(%)The fitted activity value at zero concentrationFloat%
6Sinf_(%)The fitted activity value at infinite concentrationFloat%
7Num_PointsThe number of data points used to generate the plotInteger
8Max_Activity_(%)The maximum activity value observed, based on mean of replicates per concentrationFloat%
9Max_Activity_Conc_uMThe concentration at which the maximum activity is observedFloatμM
10Max_Concentration_uMMaximum valid test concentrationFloatμM
11Activity_at_0.018uM_(%) (0.018μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity_at_0.056uM_(%) (0.056μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity_at_0.16uM_(%) (0.16μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity_at_0.5uM_(%) (0.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity_at_1.5uM_(%) (1.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity_at_4.6uM_(%) (4.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
17Activity_at_13.5uM_(%) (13.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity_at_38uM_(%) (38μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
19Activity_at_195uM_(%) (195μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 MH082355-01A2

Data Table (Concise)
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