Department of Chemistry, Washington University, St. Louis, MO 63130, United States.
Department of Chemistry, Washington University, St. Louis, MO 63130, United States; Department of Medicine, Washington University, School of Medicine, St. Louis, MO 63110, United States.
Redox Biol. 2018 Jun;16:381-387. doi: 10.1016/j.redox.2018.03.004. Epub 2018 Mar 9.
A growing appreciation of the metabolic artifacts of cell culture has generated heightened enthusiasm for performing metabolomics on populations of cells purified from tissues and biofluids. Fluorescence activated cell sorting, or FACS, is a widely used experimental approach to purify specific cell types from complex heterogeneous samples. Here we show that FACS introduces oxidative stress and alters the metabolic state of cells. Compared to unsorted controls, astrocytes subjected to FACS prior to metabolomic analysis showed altered ratios of GSSG to GSH, NADPH to NADP, and NAD to NADH. Additionally, a 50% increase in reactive oxygen species was observed in astrocytes subjected to FACS relative to unsorted controls. At a more comprehensive scale, nearly half of the metabolomic features that we profiled by liquid chromatography/mass spectrometry were changed by at least 1.5-fold in intensity due to cell sorting. Some specific metabolites identified to have significantly altered levels as a result of cell sorting included glycogen, nucleosides, amino acids, central carbon metabolites, and acylcarnitines. Although the addition of fetal bovine serum to the cell-sorting buffer decreased oxidative stress and attenuated changes in metabolite concentrations, fetal bovine serum did not preserve the metabolic state of the cells during FACS. We conclude that, irrespective of buffer components and data-normalization strategies we examined, metabolomic results from sorted cells do not accurately reflect physiological conditions prior to sorting.
对细胞培养代谢产物的认识不断加深，促使人们热衷于对从组织和生物体液中纯化的细胞群体进行代谢组学研究。荧光激活细胞分选（FACS）是一种广泛使用的实验方法，可从复杂的异质样本中纯化特定的细胞类型。在这里，我们表明 FACS 会引入氧化应激并改变细胞的代谢状态。与未分选的对照相比，进行代谢组学分析之前经过 FACS 处理的星形胶质细胞显示出 GSSG 与 GSH、NADPH 与 NADP 和 NAD 与 NADH 的比值发生改变。此外，与未分选的对照相比，FACS 处理的星形胶质细胞中观察到活性氧增加了 50%。在更全面的范围内，我们通过液相色谱/质谱分析进行了 profiling 的代谢组学特征中，有近一半的特征由于细胞分选而至少改变了 1.5 倍的强度。由于细胞分选而导致水平明显改变的一些特定代谢物包括糖原、核苷、氨基酸、中心碳代谢物和酰基辅酶 A。尽管在细胞分选缓冲液中添加胎牛血清可以降低氧化应激并减轻代谢物浓度的变化，但胎牛血清并不能在 FACS 过程中保持细胞的代谢状态。我们得出结论，无论我们检查的缓冲液成分和数据归一化策略如何，分选细胞的代谢组学结果都不能准确反映分选前的生理状况。
