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 (PDGFRa) 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 PDGFRa inhibitors, and then tested in a pilot screen against a library of 58 known inhibitors identifying mostly PDGFRa 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.

High Content Screening as High Quality Assay for Biological Evaluation of Photosensitizers In Vitro

“…Automated imaging has been available for the last 20 years and it has significantly streamlined the process of in vitro drug screening in addition to decreasing the time needed both for high quality image collection and analysis [24], [25]. Naturally fluorescent drugs have, in the past, been evaluated by fluorescent microscopy [26], [27], [28], [29]; however, a phenotypic screen of toxicity in cells using naturally fluorescent compounds has seldom been attempted due to its inherent restrictions.…”

“…The reduction is performed by the action of mitochondrial dehydrogenases in metabolically active cells; the amount of formatazan produced is known to be directly proportional to the mitochondrial enzymatic activity and, therefore, to the number of proliferating cells in culture [22], [23], [24], [25]. This assay has been extensively used both to determine cell proliferation, viability and to determine toxicity of potential therapeutic agents [35], [36], [37].…”

Designs and Concept Reliance of a Fully Automated High-Content Screening Platform

“…7 The instrument dimensions are [654mm × 1140mm × 605mm (D x W x H)] with a weight of 50 kilograms. The INCA2000 operating software (Build Version 3.0.0.43) runs on a Windows XP operating system.…”

Domain-Based Biosensor Assay to Screen for Epidermal Growth Factor Receptor Modulators in Live Cells

“…Images of cells in 384-well microplates were acquired as previously described 13 by using the following two IN Cell Analyzer platforms (GE Healthcare): the automated epifluorescence IN Cell Analyzer 2000 (INCA2000) and the line-scanning confocal automated microscope IN Cell Analyzer 3000 (INCA3000). Imaging on the INCA2000 was performed at 20 · /0.45NA magnification.…”

“…Serial doubling dilutions of compounds were prepared as previously described. 13 Controls consisted of 1% DMSO (v/v) (high control) and 10 mM gefitinib in 1% DMSO (v/v) (low control). The assay was performed according to our optimized workflow ( Table 1).…”