Metabolic mapping of astrocytes and neurons in culture using stable isotopes and gas chromatography-mass spectrometry (GC-MS)

TY - JOUR

T1 - Metabolic mapping of astrocytes and neurons in culture using stable isotopes and gas chromatography-mass spectrometry (GC-MS)

AU - Walls, Anne B.

AU - Bak, Lasse K.

AU - Sonnewald, Ursula

AU - Schousboe, Arne

AU - Waagepetersen, Helle S.

PY - 2014/1/1

Y1 - 2014/1/1

N2 - 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 suffi cient 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.

AB - 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 suffi cient 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.

KW - C glucose/glutamate/glutamine

KW - N glutamate/glutamine

KW - Astrocytes

KW - Calculations of isotopic enrichment

KW - Co-cultures

KW - Glycogen

KW - Mass spectrometry

KW - Neurons

KW - Pentose phosphate pathway

UR - http://www.scopus.com/inward/record.url?scp=84921832164&partnerID=8YFLogxK

U2 - 10.1007/978-1-4939-1059-5_4

DO - 10.1007/978-1-4939-1059-5_4

M3 - Journal article

AN - SCOPUS:84921832164

SN - 0893-2336

VL - 90

SP - 73

EP - 105

JO - Neuromethods

JF - Neuromethods

ER -