Establishment and optimization of NMR-based cell metabonomics study protocols for neonatal Sprague-Dawley rat cardiomyocytes

A quenching, harvesting, and extraction protocol was optimized for cardiomyocytes NMR metabonomics analysis in this study. Trypsin treatment and direct scraping cells in acetonitrile were compared for sample harvesting. The results showed trypsin treatment cause normalized concentration increasing o...

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Published inAnalytical biochemistry Vol. 517; pp. 50 - 52
Main Authors Zhang, Ming, Sun, Bo, Zhang, Qi, Gao, Rong, Liu, Qiao, Dong, Fangting, Fang, Haiqin, Peng, Shuangqing, Li, Famei, Yan, Xianzhong
Format Journal Article
LanguageEnglish
Published United States Elsevier Inc 15.01.2017
Subjects
Animals
Cardiomyocytes
Cell Membrane - metabolism
Cell metabonomics
Cellular metabolites extract
Magnetic Resonance Spectroscopy - methods
Metabolome - physiology
Metabolomics - methods
Myocytes, Cardiac - metabolism
NMR
Rats
Rats, Sprague-Dawley
Cardiomyocytes
Cellular metabolites extract
NMR
Cell metabonomics
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Summary:A quenching, harvesting, and extraction protocol was optimized for cardiomyocytes NMR metabonomics analysis in this study. Trypsin treatment and direct scraping cells in acetonitrile were compared for sample harvesting. The results showed trypsin treatment cause normalized concentration increasing of phosphocholine and metabolites leakage, since the trypsin-induced membrane broken and long term harvesting procedures. Then the intracellular metabolite extraction efficiency of methanol and acetonitrile were compared. As a result, washing twice with phosphate buffer, direct scraping cells and extracting with acetonitrile were chosen to prepare cardiomyocytes extracts samples for metabonomics studies. This optimized protocol is rapid, effective, and exhibits greater metabolite retention.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2016.11.009