Metabolic cytometry. Glycosphingolipid metabolism in single cells
Metabolic cytometry. Glycosphingolipid metabolism in single cells
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DOI：
10.1021/ac070716d
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发表时间：
2007-07-15
影响因子：
7.4
通讯作者：
Dovichi, Norman J.
中科院分区：
文献类型：
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作者：
Whitmore, Colin D.;Hindsgaul, Ole;Dovichi, Norman J.
鞘糖脂存在于所有脊椎动物细胞中，构成所有神经细胞的主要细胞表面决定因素，有助于细胞多样性和功能的发挥。我们报告了一种分析单细胞中鞘糖脂代谢的方法。神经节苷脂G(M1)用荧光染料四甲基罗丹明标记。这种标记的化合物被垂体瘤(ATT-20)细胞培养摄取和代谢。50h后，福尔马林固定细胞。细胞被吸入熔融石英毛细管并裂解，用激光诱导荧光检测器进行毛细管电泳法分离成分。所有保留荧光染料的代谢产物都可以在低水平的齐托莫尔检测到。使用该程序对54个ATT-20细胞进行了单独分析。电泳谱的重现性非常好，这有助于根据荧光标记标准的迁移时间来鉴定组分。共检测到11种组分，这些组分的平均峰高跨越2个数量级以上，因此可以在组分丰富的情况下检测到痕量代谢物。最高丰度组分的归一化丰度相对标准偏差为10%。平均每个细胞占据约2amol(10(6)个拷贝)的标记底物。这种方法可以确定细胞间的多样性和调节神经鞘糖脂的代谢。
Glycosphingolipids are found on all vertebrate cells and constitute major cell surface determinants on all nerve cells, where they contribute to cellular diversity and function. We report a method for the analysis of glycosphingolipid metabolism in single cells. The ganglioside G(M1) was tagged with the fluorescent dye tetramethylrhodamine. This labeled compound was taken up and metabolized by a culture of pituitary tumor (AtT-20) cells. After 50 h, the cells were formalin fixed. Cells were aspirated into a fused-silica capillary and lysed, and components were separated by capillary electrophoresis with a laser-induced fluorescence detector. All metabolic products that retained the fluorescent dye could be detected at the low-zeptomole level. A total of 54 AtT-20 cells were individually analyzed using this procedure. The electrophoretic profiles were remarkably reproducible, which facilitated identification of components based on the migration time of fluorescently labeled standards. Eleven components were detected, and the average peak height of these components spanned more than 2 orders of magnitude, so that trace metabolites can be detected in the presence of abundant components. The most highly abundant components generated 10% relative standard deviation in normalized abundance. The average cell took up roughly 2 amol (10(6) copies) of the labeled substrate. This method allows determination of cell-to-cell diversity and regulation of glycosphingolipid metabolism.