Comprehensive method for culturing embryonic dorsal root ganglion neurons for Seahorse Extracellular Flux XF24 analysis

Miranda Lange, Yan Zeng, Andrew Knight, Anthony Windebank, Eugenia Trushina

Research output: Contribution to journalArticle

7 Scopus citations

Abstract

Changes in mitochondrial dynamics and function contribute to progression of multiple neu- rodegenerative diseases including peripheral neuropathies.The Seahorse Extracellular Flux XF24 analyzer provides a comprehensive assessment of the relative state of glycolytic and aerobic metabolism in live cells making this method instrumental in assessing mitochon- drial function. One of the most important steps in the analysis of mitochondrial respiration using the Seahorse XF24 analyzer is plating a uniform monolayer of firmly attached cells. However, culturing of primary dorsal root ganglion (DRG) neurons is associated with multi- ple challenges, including their propensity to form clumps and detach from the culture plate. This could significantly interfere with proper analysis and interpretation of data. We have tested multiple cell culture parameters including coating substrates, culture medium, XF24 microplate plastics, and plating techniques in order to optimize plating conditions. Here we describe a highly reproducible method to obtain neuron-enriched monolayers of securely attached dissociated primary embryonic (E15) rat DRG neurons suitable for analysis with the Seahorse XF24 platform.

Original languageEnglish (US)
Article numberArticle 175
JournalFrontiers in Neurology
Volume3 DEC
DOIs
StatePublished - 2012

Keywords

  • Embryonic dorsal root ganglion neurons
  • Extracellular acidification rate
  • Mitochondrial respiration
  • Oxygen consumption rate
  • Seahorse XF24 Extracellular Flux analysis

ASJC Scopus subject areas

  • Neurology
  • Clinical Neurology

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