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BioAssay: AID 450

GR-GFP Redistribution

The glucocorticoid receptor (GR) Redistribution assay (BioImage) enables the visualization of GR cytoplasmic to nuclear translocation by the use of a GR-GFP fusion. GR is normally cytosolic, however ligands such as dexamethasone, cause nuclear translocation where the protein binds to response elements and interacts with various co-factors to modulate transcription. Because both functional more ..
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 Tested Compounds
 Tested Compounds
All(9966)
 
 
Active(114)
 
 
Inactive(9731)
 
 
Inconclusive(156)
 
 
 Tested Substances
 Tested Substances
All(10949)
 
 
Active(118)
 
 
Inactive(10671)
 
 
Inconclusive(160)
 
 
 Related BioAssays
 Related BioAssays
AID: 450
Data Source: NCGC (grgfp)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2006-07-18
Modify Date: 2006-11-17

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 114
Description:
NIH Molecular Libraries Screening Centers Network [MLSCN]
NIH Chemical Genomics Center [NCGC]

NCGC Assay Overview:
The glucocorticoid receptor (GR) Redistribution assay (BioImage) enables the visualization of GR cytoplasmic to nuclear translocation by the use of a GR-GFP fusion. GR is normally cytosolic, however ligands such as dexamethasone, cause nuclear translocation where the protein binds to response elements and interacts with various co-factors to modulate transcription. Because both functional agonists and antagonists can induce nuclear translocation, this assay can detect ligands regardless of their effects on gene expression. GR-GFP expressing U20S cells were used here. Dexamethasone was the positive control for translocation and data normalization. Detection of nuclear translocation was performed in 1536-well plates using the Acumen laser scanning plate cytometer [1] (TTP LabTech). Validation of this assay and a detailed protocol have been described [2].
Protocol
NCGC Assay Protocol Summary:

GR-GFP U2OS cells were maintained in DMEM with Glutamax and high glucose, 1% v/v penicillin-streptomycin, and 0.5 mg/mL Geneticin. For cell culture, 10% heat-denatured fetal bovine serum (FBS) was used (Invitrogen #26140-079). For cell plating 10% dextran, charcoal-stripped FBS (Hyclone SH30068.03) was used. U2OS cells were trypsin treated, suspended in medium, and passed through a 40 um basket filter. The cells were seeded at 600 cells/5 uL/well into clear-bottom black sided 1536-well plates and incubated for approximately 20 hrs, 37degC, 5%, CO2 in a tissue culture incubator. Twenty-three nL of compound was delivered by pin tool and the plates were incubated as above for 2 hrs. The compounds were tested as a 7-point titration ranging from 46 uM to 0.6 nM. The cells were fixed using the Kalypsys Washer/Dispenser station. The fixing protocol involved aspiration of the cell media, addition of 6 uL/well of PBS, aspiration of PBS, addition of 5uL/well of 100% methanol, aspiration of methanol, and addition of 3 uL/well PBS with 1.5 uM propidium iodide (PI). The plates were read by the Acumen Explorer using a 1 x 8 um scan of the whole well area. PI fluorescence triggered the collection of PI and GFP signals. A nuclei population was defined by a 10 to 100 um width and depth filter that eliminated small and large fluorescent particles. Two subpopulations of nuclei were then defined by peak intensity of GFP fluorescence to obtain translocated and untranslocated objects. The number of translocated objects was used for data normalization.

Keywords: NIH Roadmap, MLSCN, MLI, MLSMR, sOGT, qHTS, NCGC
Comment
References:

1. Bowen, W.P. and Wylie, P.G., "Application of Laser-Scanning Fluorescence Microplate Cytometry in High Content Screening". ASSAY Drug Devel.Technol. 2006, 4(2), 209-221

2. Auld, DS, et al., "Fluorescent Protein-based cellular assays analyzed by laser scanning microplate cytometry in 1536-well plate format" Methods in Enzymol.: Measuring Biological Responses with Automated Microscopy. Vol 414, in press.

Compound Ranking:

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive

2. Within each curve class, compounds are ranked by potency
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Activity DirectionIndicates direction of activity: inactive, decreasing, increasing.String
2Activity QualifierIndicates if AC50 is "=" to value or if it is infinite activity (i.e, inactive), ">".String
3Qualified AC50Qualified AC50; can be an IC50 or EC50.Float
4Log of AC50Log of AC50.Float
5Hill CoefficientHill slope of fitted curve.Float
6Curve R2R^2 fit value of curve. Closer to 1.0 equates to better Hill equation fit.Float
7Data TypeNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String
8Compound QCNCGC comment on compound QC: 'DPI QC', 'NCGC Purified'String
9Data Analysis QCNCGC comment on confidence of data: 'Curve Verfied', etcString
10NCGC CommentAnnotation/notes on a particular compound's data: e.g, 'cytotoxic', 'fluorescent artifact'String
11Curve Fit ModelCurve fitting model used.String
12Hill S0S0 of Hill EquationFloat
13Hill SinfSinf of Hill EquationFloat
14Hill dSdS of Hill EquationFloat
15Log AC50 Std ErrorStandard error for Log AC50.Float
16Excluded PointsIndividual points excluded from curve fitting.String
17Number of PointsNumber of valid points used for fitting.Integer
18Activity at 0.59nM% Activity at given concentration.Float%
19Activity at 1.319nM% Activity at given concentration.Float%
20Activity at 2.95nM% Activity at given concentration.Float%
21Activity at 6.597nM% Activity at given concentration.Float%
22Activity at 14.751nM% Activity at given concentration.Float%
23Activity at 0.033uM% Activity at given concentration.Float%
24Activity at 0.074uM% Activity at given concentration.Float%
25Activity at 0.165uM% Activity at given concentration.Float%
26Activity at 0.369uM% Activity at given concentration.Float%
27Activity at 0.824uM% Activity at given concentration.Float%
28Activity at 1.844uM% Activity at given concentration.Float%
29Activity at 4.122uM% Activity at given concentration.Float%
30Activity at 9.217uM% Activity at given concentration.Float%
31Activity at 20.61uM% Activity at given concentration.Float%
32Activity at 0.046mM% Activity at given concentration.Float%
33Compound TypeNCGC designation for compound stage: 'qHTS ECL (Exploratory)', 'NIHSMR (MLSCN)', 'Compound Followup', 'Compound Verification', 'Probe Optimization'String

Data Table (Concise)
Classification
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