Single-cell analysis avoids sample processing bias
Single-cell analysis avoids sample processing bias
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DOI：
10.1016/s0378-4347(99)00539-3
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发表时间：
2000-04-28
影响因子：
3
通讯作者：
Dovichi, NJ
中科院分区：
文献类型：
--
作者：
Krylov, SN;Arriaga, E;Dovichi, NJ
毛细管色谱和毛细管电泳 (CE) 等微型分离工具可用于研究单个细胞的代谢。在这项工作中，我们证明单细胞分析比细胞提取物分析更准确地描述新陈代谢。我们用荧光标记的代谢探针孵育 HT29 细胞（人结肠腺癌）。这种二糖 LacNAc 用荧光染料四甲基罗丹明 (TMR) 进行标记。探针被细胞吸收并代谢成许多保留荧光标记的产物。然后我们将细胞分成两批。从一批中制备细胞提取物，并通过 CE 和激光诱导荧光 (LIF) 检测进行分析。第二批细胞用于通过 CE-LIF 进行单细胞分析。提取物和单细胞分析的分离和检测条件相同。我们发现，通过对多个单细胞的结果进行平均而获得的电泳图与细胞提取物电泳图有显着差异。差异是由于提取物制备过程中的样品处理造成的。细胞的破坏会释放出细胞内的酶，从而使非代谢反应得以进行。这些非代谢产物的积累在细胞提取物测定中引入了偏差。在单细胞分析过程中，细胞在毛细管内裂解，并立即施加分离电压以将酶与其底物分离并防止非代谢反应。本文首次报道了单细胞的 CE 分析比细胞提取物的 CE 分析提供了更准确的代谢信息。 (C) 2000 Elsevier Science B.V. 保留所有权利。
Microscale separation tools such as capillary chromatography and capillary electrophoresis (CE) allow the study of metabolism in individual cells. In this work, we demonstrate that single-cell analysis describes metabolism more accurately than analysis of cellular extracts. We incubated HT29 cells (human colon adenocarcinoma) with a fluorescently labeled metabolic probe. This disaccharide, LacNAc, was labeled with a fluorescent dye, tetramethylrhodamine (TMR). The probe was taken up by the cells and metabolized to a number of products that retained the fluorescent label. We then split the cells into two batches. A cellular extract was prepared from one batch and analyzed by CE with laser-induced fluorescence (LIF) detection. The cells from the second batch were used for single-cell analysis by CE-LIF. Separation and detection conditions were identical for extract and single-cell analyses. We found that the electropherogram obtained by averaging the results from a number of single cells differed significantly from the cell extract electropherogram. Differences were due to sample processing during extract preparation. Disruption of the cells liberated enzymes that were compartmentalized within the cell, which allowed non-metabolic reactions to proceed. The accumulation of these non-metabolic products introduced a bias in the cell extract assay. During single-cell analysis, cells were lysed inside the capillary and the separation voltage was applied immediately to separate the enzymes from their substrates and prevent non-metabolic reactions. This paper is the first to report that CE analysis of single cells provides more accurate metabolic information than the CE analysis of a cellular extract. (C) 2000 Elsevier Science B.V. All rights reserved.