Determination of the time course and extent of neurotoxicity at defined temperatures in cultured neurons using a modified multiwell plate fluorescence scanner

Rita Sattler, Milton P. Charlton, Mathias Hafner, Michael Tymianski

Research output: Contribution to journalArticle

Abstract

The cellular and molecular mechanisms of hypoxic/ischemic neurodegeneration are sensitive to numerous factors that modulate the time course and degree of neuronal death. Among such factors is hypothermia, which can dramatically protect neurons from injury. To examine and control for temperature-dependent effects, we developed a technique that provides for a high-throughput, accurate, and reproducible determination of the time course and degree of neurotoxicity in cultured cortical neurons at precisely defined temperatures. We used a fluorescence multiwell plate scanner, modified by us to permit the control of temperature, to perform serial quantitative measurements of propidium iodide (PI) fluorescence in cortical neuronal cultures exposed to excitotoxic insults. In validating this approach, we show that these time course measurements correlate highly with manual counts of PI-stained cells in the same cultures (r = 0.958. p < 0.0001) and with lactate dehydrogenase release (r = 0.964, p < 0.0001). This method represents an efficient approach to mechanistic and quantitative studies of cell death as well as a high-throughput technique for screening new neuroprotective therapies in vitro.

Original languageEnglish (US)
Pages (from-to)455-463
Number of pages9
JournalJournal of Cerebral Blood Flow and Metabolism
Volume17
Issue number4
DOIs
StatePublished - Apr 1997

Keywords

  • Cell death
  • Cytotoxicity assay
  • Excitotoxicity
  • Hypothermia
  • Neurotoxicity
  • Propidium iodide

ASJC Scopus subject areas

  • Neurology
  • Clinical Neurology
  • Cardiology and Cardiovascular Medicine

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