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Validation of a high-content screening assay using whole-well imaging of transformed phenotypes.

ABSTRACT: Automated microscopy was introduced two decades ago and has become an integral part of the discovery process as a high-content screening platform with noticeable challenges in executing cell-based assays. It would be of interest to use it to screen for reversers of a transformed cell phenotype. In this report, we present data obtained from an optimized assay that identifies compounds that reverse a transformed phenotype induced in NIH-3T3 cells by expressing a novel oncogene, KP, resulting from fusion between platelet derived growth factor receptor alpha (PDGFR?) and kinase insert domain receptor (KDR), that was identified in human glioblastoma. Initial image acquisitions using multiple tiles per well were found to be insufficient as to accurately image and quantify the clusters; whole-well imaging, performed on the IN Cell Analyzer 2000, while still two-dimensional imaging, was found to accurately image and quantify clusters, due largely to the inherent variability of their size and well location. The resulting assay exhibited a Z' value of 0.79 and a signal-to-noise ratio of 15, and it was validated against known effectors and shown to identify only PDGFR? inhibitors, and then tested in a pilot screen against a library of 58 known inhibitors identifying mostly PDGFR? inhibitors as reversers of the KP induced transformed phenotype. In conclusion, our optimized and validated assay using whole-well imaging is robust and sensitive in identifying compounds that reverse the transformed phenotype induced by KP with a broader applicability to other cell-based assays that are challenging in HTS against chemical and RNAi libraries.

SUBMITTER: Ramirez CN

PROVIDER: S-EPMC3123874 | biostudies-other | 2011 Jun

REPOSITORIES: biostudies-other

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Validation of a high-content screening assay using whole-well imaging of transformed phenotypes.

Ramirez Christina N CN Ozawa Tatsuya T Takagi Toshimitsu T Antczak Christophe C Shum David D Graves Robert R Holland Eric C EC Djaballah Hakim H

Assay and drug development technologies 20101223 3

Automated microscopy was introduced two decades ago and has become an integral part of the discovery process as a high-content screening platform with noticeable challenges in executing cell-based assays. It would be of interest to use it to screen for reversers of a transformed cell phenotype. In this report, we present data obtained from an optimized assay that identifies compounds that reverse a transformed phenotype induced in NIH-3T3 cells by expressing a novel oncogene, KP, resulting from ...[more]

PMID: 21182456

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Project description:Functional imaging can provide a level of quantification that is not possible in what might be termed traditional high-content screening. This is due to the fact that the current state-of-the-art high-content screening systems take the approach of scaling-up single cell assays, and are therefore based on essentially pictorial measures as assay indicators. Such phenotypic analyses have become extremely sophisticated, advancing screening enormously, but this approach can still be somewhat subjective. We describe the development, and validation, of a prototype high-content screening platform that combines steady-state fluorescence anisotropy imaging with fluorescence lifetime imaging (FLIM). This functional approach allows objective, quantitative screening of small molecule libraries in protein-protein interaction assays. We discuss the development of the instrumentation, the process by which information on fluorescence resonance energy transfer (FRET) can be extracted from wide-field, acceptor fluorescence anisotropy imaging and cross-checking of this modality using lifetime imaging by time-correlated single-photon counting. Imaging of cells expressing protein constructs where eGFP and mRFP1 are linked with amino-acid chains of various lengths (7, 19 and 32 amino acids) shows the two methodologies to be highly correlated. We validate our approach using a small-scale inhibitor screen of a Cdc42 FRET biosensor probe expressed in epidermoid cancer cells (A431) in a 96 microwell-plate format. We also show that acceptor fluorescence anisotropy can be used to measure variations in hetero-FRET in protein-protein interactions. We demonstrate this using a screen of inhibitors of internalization of the transmembrane receptor, CXCR4. These assays enable us to demonstrate all the capabilities of the instrument, image processing and analytical techniques that have been developed. Direct correlation between acceptor anisotropy and donor FLIM is observed for FRET assays, providing an opportunity to rapidly screen proteins, interacting on the nano-meter scale, using wide-field imaging.

Dataset Information

Validation of a high-content screening assay using whole-well imaging of transformed phenotypes.

Publications

Validation of a high-content screening assay using whole-well imaging of transformed phenotypes.

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