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BioAssay: AID 488805

Hypoxia inducible factor pathway Measured in Cell-Based System Using Plate Reader - 2030-01_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
All(59)
 
 
Active(28)
 
 
Inactive(31)
 
 
 Tested Substances
 Tested Substances
All(59)
 
 
Active(28)
 
 
Inactive(31)
 
 
 Related BioAssays
 Related BioAssays
AID: 488805
Data Source: Broad Institute (2030-01_Activator_Dose_DryPowder_Activity_Set2)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-10-18
Hold-until Date: 2011-10-15
Modify Date: 2011-10-15

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 28
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
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
493234Hypoxia inducible factor pathway Zinc Measured in Cell-Based System Using Plate Reader - 2030-05_Activator_Dose_DryPowder_Activity_Set2Confirmatorysame 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:
Identification of probe(s) activating the transcription factor hypoxia inducible factor (HIF) pathway. More specifically, compounds inducing a luminescent response (RLU) greater than 10% of the signal obtained from the in-plate positive control (100 uM Desferrioxamine) added to the negative control (DMSO) background will be considered hits, though this threshold may be adjusted based on number of hits. For retest at dose using dry powders, compounds inducing a dose curve higher than 30% with an EC50 below 33 uM will be considered actives.
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 (Invitrogen, SKU# 11995) as mentioned above (for normal cell propagation) or DMEM medium without phenol (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 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 (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. 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. 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) and positive control wells (PC) 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 'Stimulators Minus Neutral Controls' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of 100.
Experimental wells values were scaled to this range.


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


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.
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_0.018uM (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%

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

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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