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


Authors: Ramirez, C. N.; Ozawa, T.; Takagi, T.; Antczak, C.; Shum, D.; Graves, R.; Holland, E. C.; Djaballah, H.
Article Title: 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 (PDGFRα) 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 PDGFRα inhibitors, and then tested in a pilot screen against a library of 58 known inhibitors identifying mostly PDGFRα 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. © 2011, Mary Ann Liebert, Inc.
Journal Title: Assay and Drug Development Technologies
Volume: 9
Issue: 3
ISSN: 1540-658X
Publisher: Mary Ann Liebert, Inc  
Date Published: 2011-06-01
Start Page: 247
End Page: 261
Language: English
DOI: 10.1089/adt.2010.0342
PROVIDER: scopus
PMCID: PMC3123874
PUBMED: 21182456
DOI/URL:
Notes: --- - "Export Date: 23 June 2011" - "CODEN: ADDTA" - "Source: Scopus"
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MSK Authors
  1. Eric Holland
    225 Holland
  2. Toshimitsu Takagi
    6 Takagi
  3. Hakim Djaballah
    101 Djaballah
  4. Christophe Antczak
    40 Antczak
  5. Tatsuya Ozawa
    16 Ozawa
  6. David Shum
    54 Shum
  7. Christina Nicole Ramirez
    10 Ramirez