An imaging assay to analyze primary neurons for cellular neurotoxicity

J Neurosci Methods. 2010 Sep 30;192(1):7-16. doi: 10.1016/j.jneumeth.2010.07.003. Epub 2010 Jul 8.

Abstract

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.

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Animals
  • Animals, Newborn
  • Antigens / metabolism
  • Astrocytes / drug effects
  • Astrocytes / metabolism
  • Bungarotoxins / toxicity
  • Cell Count / methods
  • Cells, Cultured
  • Cerebellum / cytology
  • Dimethyl Sulfoxide / pharmacology
  • Dose-Response Relationship, Drug
  • Humans
  • Inhibitory Concentration 50
  • Microscopy, Confocal / methods*
  • Nerve Net / drug effects
  • Neurites / drug effects
  • Neuroblastoma / pathology
  • Neurons / cytology*
  • Neurons / drug effects*
  • Neurons / metabolism
  • Neurotoxins / pharmacology*
  • O Antigens / metabolism
  • Oligodendroglia / drug effects
  • Oligodendroglia / metabolism
  • Proteoglycans / metabolism
  • Rats
  • Time Factors
  • Tubulin / metabolism

Substances

  • Antigens
  • Bungarotoxins
  • Neurotoxins
  • O Antigens
  • Proteoglycans
  • Tubulin
  • chondroitin sulfate proteoglycan 4
  • Adenosine Triphosphate
  • Dimethyl Sulfoxide