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].

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

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