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Modified Protocol of Harvesting, Extraction, and Normalization Approaches for Gas Chromatography Mass Spectrometry-Based Metabolomics Analysis of Adherent Cells Grown Under High Fetal Calf Serum Conditions.
Metabolites ( IF 3.4 ) Pub Date : 2019-12-18 , DOI: 10.3390/metabo10010002
Raphaela Fritsche-Guenther 1, 2 , Anna Bauer 1, 2 , Yoann Gloaguen 1, 2, 3 , Mario Lorenz 4, 5 , Jennifer A Kirwan 1, 2
Affiliation  

A gas chromatography mass spectrometry (GC-MS) metabolomics protocol was modified for quenching, harvesting, and extraction of metabolites from adherent cells grown under high (20%) fetal calf serum conditions. The reproducibility of using either 50% or 80% methanol for quenching of cells was compared for sample harvest. To investigate the efficiency and reproducibility of intracellular metabolite extraction, different volumes and ratios of chloroform were tested. Additionally, we compared the use of total protein amount versus cell mass as normalization parameters. We demonstrate that the method involving 50% methanol as quenching buffer followed by an extraction step using an equal ratio of methanol:chloroform:water (1:1:1, v/v/v) followed by the collection of 6 mL polar phase for GC-MS measurement was superior to the other methods tested. Especially for large sample sets, its comparative ease of measurement leads us to recommend normalization to protein amount for the investigation of intracellular metabolites of adherent human cells grown under high (or standard) fetal calf serum conditions. To avoid bias, care should be taken beforehand to ensure that the ratio of total protein to cell number are consistent among the groups tested. For this reason, it may not be suitable where culture conditions or cell types have very different protein outputs (e.g., hypoxia vs. normoxia). The full modified protocol is available in the Supplementary Materials.

中文翻译:

在高胎牛血清条件下生长的贴壁细胞的基于气相色谱质谱的代谢组学分析的收获,提取和归一化方法的改进方案。

修改了气相色谱质谱(GC-MS)代谢组学方案,以淬灭,收集和提取胎牛血清浓度高(20%)的贴壁细胞中的代谢物。比较了使用50%或80%甲醇淬灭细胞的可重复性,以进行样品收集。为了研究细胞内代谢产物提取的效率和可重复性,测试了不同体积和比例的氯仿。此外,我们比较了使用总蛋白量与细胞量作为归一化参数的情况。我们证明了该方法涉及50%甲醇作为淬灭缓冲液,然后使用等比例的甲醇:氯仿:水(1:1:1,v / v / v)进行萃取,然后收集6 mL极性相GC-MS测量优于其他测试方法。尤其是对于大型样品,其相对容易测量,因此我们建议对蛋白质量进行归一化,以研究在高(或标准)胎牛血清条件下生长的粘附人类细胞的细胞内代谢产物。为避免产生偏见,应事先注意确保测试组中总蛋白与细胞数的比例一致。因此,在培养条件或细胞类型具有非常不同的蛋白质输出(例如,低氧与正常氧)的情况下,可能不适合。完整的修改后的协议可在补充材料中找到。它比较容易测量,因此我们建议对蛋白质量进行归一化,以研究在高(或标准)胎牛血清条件下生长的粘附人类细胞的细胞内代谢产物。为避免偏倚,应事先注意确保测试组中总蛋白与细胞数的比例一致。因此,在培养条件或细胞类型具有非常不同的蛋白质输出(例如,低氧与正常氧)的情况下,可能不适合。完整的修改后的协议可在补充材料中找到。它比较容易测量,因此我们建议对蛋白质量进行归一化处理,以研究在高(或标准)胎牛血清条件下生长的粘附人类细胞的细胞内代谢产物。为避免产生偏见,应事先注意确保测试组中总蛋白与细胞数的比例一致。因此,在培养条件或细胞类型具有非常不同的蛋白质输出(例如,低氧与正常氧)的情况下,可能不适合。完整的修改后的协议可在补充材料中找到。如果培养条件或细胞类型具有非常不同的蛋白质输出(例如,低氧与正常氧),则可能不适合。完整的修改后的协议可在补充材料中找到。如果培养条件或细胞类型具有非常不同的蛋白质输出(例如,低氧与正常氧),则可能不适合。完整的修改后的协议可在补充材料中找到。
更新日期:2019-12-19
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