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Fast and ergonomic extraction of adherent mammalian cells for NMR-based metabolomics studies.
Analytical and Bioanalytical Chemistry ( IF 3.8 ) Pub Date : 2020-06-18 , DOI: 10.1007/s00216-020-02764-9
Manhal Mili 1 , Baptiste Panthu 2 , Anne-Marie Madec 2 , Marie-Agnès Berger 2 , Gilles J P Rautureau 1 , Bénédicte Elena-Herrmann 3
Affiliation  

Cellular metabolomics has become key to elucidate mechanistic aspects in various fields such as cancerology or pharmacology, and is rapidly becoming a standard phenotyping tool accessible to the broad biological community. Acquisition of reliable spectroscopic datasets, such as nuclear magnetic resonance (NMR) spectra, to characterize biological systems depends on the elaboration of robust methods for cellular metabolites extraction. Previous studies have addressed many issues raised by these protocols, however with little pondering on ergonomic and practical aspects of the methods that impact their scalability, reproducibility and hence their suitability to high-throughput studies or their use by non-metabolomics experts. Here, we optimize a fast and ergonomic protocol for extraction of metabolites from adherent mammalian cells for NMR metabolomics studies. The proposed extraction protocol, including cell washing, metabolism quenching and actual extraction of intracellular metabolites, was first optimized on HeLa cells. Efficiency of the protocol, in its globality and for the different individual steps, was assessed by NMR quantification of 27 metabolites from cellular extracts. We show that a single PBS wash provides a seemly compromise between contamination from growth medium and leakage of intracellular metabolites. In HeLa cells, extraction using pure methanol, without cell scraping, recovered a higher amount of intracellular metabolites than the reference methanol/water/chloroform method with cell scraping, with yields varying across metabolite classes. Optimized and reference protocols were further tested on eight cell lines of miscellaneous nature, and inter-operator reproducibility was demonstrated. Our results stress the need for tailored extraction protocols and show that fast protocols minimizing time-consuming steps, without compromising extraction yields, are suitable for high-throughput metabolomics studies.

Graphical abstract



中文翻译:

快速,符合人体工程学的贴壁哺乳动物细胞提取,用于基于NMR的代谢组学研究。

细胞代谢组学已成为阐明各种领域(例如癌症学或药理学)机理的关键,并且正迅速成为广泛的生物学界可访问的标准表型分析工具。获得可靠的光谱数据集(例如核磁共振(NMR)光谱)以表征生物系统取决于对细胞代谢物提取的可靠方法的详细描述。先前的研究已经解决了这些协议提出的许多问题,但是很少考虑方法的人体工程学和实践方面会影响其可扩展性,可重复性,从而影响其对高通量研究的适应性或非代谢组学专家的使用。这里,我们优化了一种快速且符合人体工程学的方案,用于从附着的哺乳动物细胞中提取代谢物用于NMR代谢组学研究。首先在HeLa细胞上优化了拟议的提取方案,包括细胞洗涤,代谢猝灭和细胞内代谢物的实际提取。通过NMR定量分析细胞提取物中27种代谢产物,评估了该协议在全局性和不同步骤中的有效性。我们显示,单次PBS洗涤在生长培养基的污染与细胞内代谢物的泄漏之间似乎提供了一种折衷方案。在HeLa细胞中,使用纯甲醇进行提取而不进行细胞刮除,比使用细胞刮除的参考甲醇/水/氯仿方法回收的细胞内代谢物的回收量更高,且代谢物的种类不同。优化方案和参考方案在其他八种细胞系中进行了进一步测试,并证明了操作员之间的可重复性。我们的结果强调了对量身定制的提取方案的需求,并表明快速的方案可将耗时的步骤减至最少,而不会影响提取的收率,适用于高通量代谢组学研究。

图形概要

更新日期:2020-06-18
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