Sorting cells alters their redox state and cellular metabolome.
Sorting cells alters their redox state and cellular metabolome.
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DOI：
10.1016/j.redox.2018.03.004
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发表时间：
2018-06
期刊：
影响因子：
11.4
通讯作者：
Patti GJ
中科院分区：
文献类型：
--
作者：
Llufrio EM;Wang L;Naser FJ;Patti GJ
人们对细胞培养代谢产物的认识不断加深，引发了人们对从组织和生物体液中纯化的细胞群进行代谢组学的热情高涨。荧光激活细胞分选 (FACS) 是一种广泛使用的实验方法，用于从复杂的异质样品中纯化特定细胞类型。在这里，我们证明 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，在代谢组学分析之前进行 FACS 的星形胶质细胞显示 GSSG 与 GSH、NADPH 与 NADP+ 以及 NAD+ 与 NADH 的比率发生了变化。此外，相对于未分选的对照，在接受 FACS 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/质谱分析的代谢组学特征中近一半的强度由于细胞分选而改变了至少 1.5 倍。由于细胞分选，一些特定的代谢物被鉴定为水平显着改变，包括糖原、核苷、氨基酸、中心碳代谢物和酰基肉碱。尽管在细胞分选缓冲液中添加胎牛血清可降低氧化应激并减弱代谢物浓度的变化，但胎牛血清在 FACS 过程中并不能保持细胞的代谢状态。我们的结论是，无论我们检查的缓冲液成分和数据标准化策略如何，分选细胞的代谢组学结果都不能准确反映分选前的生理条件。让细胞接受 FACS 会引入氧化应激并改变细胞氧化还原状态。许多代谢物的浓度在细胞分选过程中发生变化。 FBS 和 BSA 不能防止 FACS 期间的这些干扰。代谢变化是不均匀的，不能通过简单的标准化来纠正。
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. Subjecting cells to FACS introduces oxidative stress and alters cellular redox state. The concentrations of many metabolites change during cell sorting. FBS and BSA do not prevent these perturbations during FACS. Metabolic changes are non-uniform and cannot be corrected by simple normalization.
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影响因子：
7.4
作者：
Mahieu NG;Patti GJ
通讯作者：
Patti GJ
影响因子：
8.8
作者：
Gelman SJ;Naser F;Mahieu NG;McKenzie LD;Dunn GP;Chheda MG;Patti GJ
通讯作者：
Patti GJ
影响因子：
1.6
作者：
BONNER, WA;SWEET, RG;HERZENBERG, LA
通讯作者：
HERZENBERG, LA
影响因子：
3.5
作者：
ATTRAMADAL A
通讯作者：
ATTRAMADAL A
影响因子：
11.2
作者：
Kamphorst JJ;Nofal M;Commisso C;Hackett SR;Lu W;Grabocka E;Vander Heiden MG;Miller G;Drebin JA;Bar-Sagi D;Thompson CB;Rabinowitz JD
通讯作者：
Rabinowitz JD