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BioAssay: AID 492962

Mouse Th1 T cell differentiation assay for inhibitors of ROR gamma transcriptional activity

Keywords: NIH Roadmap, MLPCN, MLI, MLSMR, qHTS, NCGC, qHTS, nuclear hormone receptor, retinoic acid-related orphan receptor, ROR gamma ..more
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 Tested Compounds
 Tested Compounds
All(3)
 
 
Inactive(3)
 
 
 Tested Substances
 Tested Substances
All(3)
 
 
Inactive(3)
 
 
AID: 492962
Data Source: NCGC (RORG529)
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-12-06
Hold-until Date: 2011-12-01
Modify Date: 2011-12-02

Data Table ( Complete ):           View All Data
Target
Tested Compounds:
Related Experiments
Show more
AIDNameTypeComment
2551qHTS for inhibitors of ROR gamma transcriptional activityConfirmatorydepositor-specified cross reference
2604Quantitative high throughput screen for inhibitors of ROR gamma transcriptional activity: SummarySummarydepositor-specified cross reference
2546VP16 counterscreen qHTS for inhibitors of ROR gamma transcriptional activityConfirmatorysame project related to Summary assay
2762Concentration response confirmation assay for inhibitors of ROR gamma transcriptional activityConfirmatorysame project related to Summary assay
2763Concentration response confirmation VP16 counterscreen for inhibitors of ROR gamma transcriptional activityConfirmatorysame project related to Summary assay
489036VP16 selectivity assay for inhibitors of ROR gamma transcriptional activityConfirmatorysame project related to Summary assay
489037Secondary confirmation assay for inhibitors of ROR gamma transcriptional activityConfirmatorysame project related to Summary assay
489038DHR3 selectivity assay for inhibitors of ROR gamma transcriptional activityConfirmatorysame project related to Summary assay
489039ROR alpha selectivity assay for inhibitors of ROR gamma transcriptional activityConfirmatorysame project related to Summary assay
492954Mouse Th17 T cell differentiation assay for inhibitors of ROR gamma transcriptional activityConfirmatorysame project related to Summary assay
Description:
NIH Molecular Libraries Probe Production Network [MLPCN]
NIH Chemical Genomics Center [NCGC]

MLPCN Grant: R03 DA026211-01
Assay Provider: Dan Littman, New York University

Keywords: NIH Roadmap, MLPCN, MLI, MLSMR, qHTS, NCGC, qHTS, nuclear hormone receptor, retinoic acid-related orphan receptor, ROR gamma

Assay Overview:

The retinoic acid-related orphan receptor (ROR) gamma is a transcription factor that has a central role in the differentiation of Th17 cells, a subset of T helper cells that secrete the inflammatory cytokines IL-17a, IL-17f, and IL-22. Th17 cells have been implicated in graft versus host disease, autoimmune disease and asthma. ROR gamma is induced in naive T helper cells in the presence of TGF-beta combined with IL-6, IL-21, or IL-23, and thereafter directs the expression of the Th17 lineage cytokines. To confirm the selectivity of ROR gamma inhibitors, compounds with strong inhibitory activities on Th17 cells were tested in a mouse Th1 T cell differentiation assay. T cells differentiate into Th1 cells in the presence of IL-12 and IL-2. Th1 differentiation does not require ROR gamma and thus selective ROR gamma inhibitors should not block this process. Th1 differentiation was tracked by the induction of interferon gamma (IFN-g) expression as measured by an anti-IFN-g antibody using flow cytometry. Selective ROR gamma inhibitors do not block IFN-g expression in this assay.
Protocol
Assay Protocol Summary:

Cells from lymph nodes and spleens derived from six to eight week old IL17a-GFP mice (Biocytogen LLC) were used for T cell purification. B220- cells were isolated on an autoMACS Pro with bead depletion of B220+ cells (Miltenyi). Naive CD4+ T cell were further purified as TCRb+CD8- DAPI- CD19- CD4+CD25- CD62L+CD44low/Int by cell sorting on a FACSAria (BD). Cells were cultivated in an incubator at 37 C and 5% CO2 in RPMI 1640 medium (Invitrogen) supplemented with 10% (vol/vol) heat-inactivated FBS (Hyclone), penicillin-streptomycin, 2 mM glutamine and 0.1 mM nonessential amino acids. Cells were seeded on day 0 at a density of 0.4E5 cells per ml in 96-well plates coated with anti-CD3e (5 ug/ml) and anti-CD28 (10 ug/ml). Cells were cultured for 4~5 days in Th1 [IL-12 (10 ng/ml), IL-2 (100 U/ml), anti-IL-4] condition. At day 1, compounds dissolved in DMSO were added. For intracellular cytokine staining, cells were incubated for 5 h with phorbol ester (50 ng/ml; Sigma), ionomycin (500 ng/ml; Sigma) and GolgiStop (BD). When needed, surfaces were stained with PECy7-conjugated CD4 (BD Biosciences) by incubating on ice for 15 min. The Cytofix/Cytoperm buffer set (BD) was used for intracellular staining. Cells were fixed, permeabilized for 30 min on ice and stained for 30 min on ice in permeabilization buffer with Alexa647-conjugated anti-IL17a (eBioscience) and PE-conjugated anti-IFN-g (eBioscience). An LSR II (BD Biosciences) and FlowJo software (Tree Star) were used for flow cytometry.

Keywords: NIH Roadmap, MLPCN, MLI, MLSMR, qHTS, NCGC, qHTS, nuclear hormone receptor, retinoic acid-related orphan receptor, ROR gamma
Comment
Compound Ranking:

All compounds are inactive and assigned a PUBCHEM_ACTIVITY_SCORE of 0.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Cell Type: T cell
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1% Activity% Activity at test concentration.Float%
2Test concentration (uM)Concentration tested.FloatμM
3Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String
Additional Information
Grant Number: R03 DA026211-01

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