 | High Content Screening (HCS) enables the evaluation of multiple
parameters in cellular systems by combining the automated imaging of
cells with high-quality detection reagents and powerful image analysis
algorithms, representing a powerful new analysis tool for drug discovery.
We have developed a multi-parameter approach for profiling of
compound neurotoxicity using HCS technology. We have utilized a variety
of cellular models, including primary rat neurons and astrocytes, PC12
cells, neuroblastoma cells and human embryonic stem cell-derived
neuronal cells, to examine a panel of antibodies specific for a variety of
putative neurotoxicity biomarkers including neuronal damage, astrocyte
hypertrophy and gliosis. Each antibody was evaluated for antibodyantigen
specificity, signal-to-background ratio, and their ability to detect
compound neurotoxicity. For image acquisition and analysis, the GE IN
Cell Analyzer 1000 high-content imaging system was used. We
demonstrate that appropriate choice of antibodies and reagents is
important for clear identification of neurons and glia amongst a mixed cell
population. Using an optimal combination of detection reagents we were
able to perform robust screening assays for quantification of neurotoxic
responses to known toxicants, including the microtubule depolymerization
agent nocodazole and the protein kinase inhibitor K252a. Additionally, by
calculating signal-to-background ratios, we were able to demonstrate that
the assay reagents are stable for at least 24 hr at room temperature,
enabling large-scale screening applications. In conclusion, we have
developed assays that provide the opportunity to perform large-scale,
non-subjective, quantitative assays for neurotoxicity biomarkers, and
enable morphological screening of multiple parameters in individual cells.
These assays offer the opportunity of better detection of neurotoxicity and
greatly improved.
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