An imaging assay to analyze primary neurons for cellular neurotoxicity

The development of high-content screening technologies including automated immunostaining, automated image acquisition and automated image analysis have enabled higher throughput of cellular imaging-based assays. Here we used high-content imaging to thoroughly characterize the cultures of primary ra...

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Bibliographic Details
Published in:Journal of neuroscience methods 2010-09, Vol.192 (1), p.7-16
Main Authors: Götte, Marjo, Hofmann, Gabriele, Michou-Gallani, Anne-Isabelle, Glickman, J. Fraser, Wishart, William, Gabriel, Daniela
Format: Article
Language:English
Subjects:
Adenosine Triphosphate - metabolism
Animals
Animals, Newborn
Antigens - metabolism
Astrocytes - drug effects
Astrocytes - metabolism
Bungarotoxins - toxicity
Cell Count - methods
Cell culture
Cells, Cultured
Cerebellar granule neurons
Cerebellum - cytology
Dimethyl Sulfoxide - pharmacology
Dose-Response Relationship, Drug
High-content analysis
Humans
Inhibitory Concentration 50
Microscopy, Confocal - methods
Nerve Net - drug effects
Neurite outgrowth
Neurites - drug effects
Neuroblastoma - pathology
Neurons - cytology
Neurons - drug effects
Neurons - metabolism
Neurotoxicity
Neurotoxins - pharmacology
O Antigens - metabolism
Oligodendroglia - drug effects
Oligodendroglia - metabolism
Proteoglycans - metabolism
Rats
Time Factors
Tubulin - metabolism
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Summary:The development of high-content screening technologies including automated immunostaining, automated image acquisition and automated image analysis have enabled higher throughput of cellular imaging-based assays. Here we used high-content imaging to thoroughly characterize the cultures of primary rat cerebellar granule neurons (CGNs). We describe procedures to isolate and cultivate the CGNs in 96-well and 384-well format, as well as a procedure to freeze and thaw the CGNs. These methods allow the use of CGNs in 96-well format analyzing 2500 samples per experiment using freshly isolated cells. Down-scaling to 384-well format and freezing and thawing of the CGNs allow even higher throughput. A cellular assay with rat CGN cultures was established to study the neurotoxicity of compounds in order to filter out toxic compounds at an early phase of drug development. The imaging-based toxicity assay was able to reveal adverse effects of compounds on primary neurons which were not detected in neuroblastoma or other cell lines tested.
ISSN:0165-0270
1872-678X
DOI:10.1016/j.jneumeth.2010.07.003