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Metabolic Mapping of Astrocytes and Neurons in Culture Using Stable Isotopes and Gas Chromatography-Mass Spectrometry (GC-MS)

  • Anne B. WallsEmail author
  • Lasse K. Bak
  • Ursula Sonnewald
  • Arne Schousboe
  • Helle S. Waagepetersen
Protocol
Part of the Neuromethods book series (NM, volume 90)

Abstract

The experimental procedure involved in metabolic mapping of astrocytes and neurons in culture using stable isotopes and gas chromatography-mass spectrometry (GC-MS) comprises several steps each of which has to be thoroughly considered in order to obtain the desired information. In this chapter we describe the different aspects to be considered when designing an incubation experiment to provide information about cellular metabolism in vitro, i.e., incubation time, incubation medium, and which isotope to employ. The labeling patterns obtained in several metabolites following metabolism of a variety of 13C or 15N labeled precursors through different metabolic pathways are depicted in order to provide sufficient insight to enable the reader to select the best suited precursor. The cell extraction and sample preparation procedures required before GC-MS analysis are described as are the subsequent integration of chromatograms and the calculations needed to correct for natural abundance and to obtain percentage labeling of M, M + 1, M + 2, etc. in a compound. Also, the cell culturing procedure for preparing primary cultures of neurons and astrocytes and also co-cultures of these cell types isolated from either mouse cerebral cortex or cerebellum is described in detail.

Key words

Astrocytes Neurons Co-cultures 13C glucose/glutamate/glutamine 15N glutamate/glutamine Glycogen Pentose phosphate pathway Mass spectrometry Calculations of isotopic enrichment 

Abbreviations

CSF

Cerebrospinal fluid

dBcAMP

dibutyryl cyclic AMP

DMF

N,N-Dimethylformamide

f.c.

Final concentration

GABA-T

GABA transaminase

GAD

Glutamate decarboxylase

GC-MS

Gas chromatography-mass spectrometry

GDH

Glutamate dehydrogenase

GS

Glutamine synthetase

LAF hood

Laminar flow hood

MCL

Molecular carbon labeling

MS

Mass spectrometry

MSTFA

N-methyl-N-(trimethylsilyl)trifluoroacetamide

MTBSTFA

N-methyl-N-(tert.-butyldimethylsilyl)trifluoroactamide

NMR

Nuclear magnetic resonance

PC

Pyruvate carboxylase

PDH

Pyruvate dehydrogenase

PEPCK

Phosphoenolpyruvate carboxykinase

PPP

Pentose phosphate pathway

TCA

Tricarboxylic acid

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Anne B. Walls
    • 1
    Email author
  • Lasse K. Bak
    • 1
  • Ursula Sonnewald
    • 2
  • Arne Schousboe
    • 1
  • Helle S. Waagepetersen
    • 3
  1. 1.Department of Drug Design and Pharmacology, Faculty of Health and Medical SciencesUniversity of CopenhagenCopenhagenDenmark
  2. 2.Department of Neuroscience, Faculty of MedicineNorwegian University of Science and TechnologyTrondheimNorway
  3. 3.Faculty of Health and Medical SciencesUniversity of CopenhagenCopenhagenDenmark

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