Analysis of mitochondria isolated from single cells
Analysis of mitochondria isolated from single cells
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DOI：
10.1007/s00216-006-0689-6
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发表时间：
2007-01-01
影响因子：
4.3
通讯作者：
Arriaga, Edgar A.
中科院分区：
文献类型：
--
作者：
Johnson, Ryan D.;Navratil, Marian;Arriaga, Edgar A.
批量研究不适合描述和研究细胞间变异，这在生物过程中非常重要，如胚胎发生、组织分化和疾病。以前，毛细管电泳与激光诱导荧光检测（CE-LIF）用于测量从数百万细胞中分离的细胞器的特性。因此，这些批量测量报告了细胞群细胞器的平均性质。从单细胞释放的细胞器的类似测量将是高度相关的，以描述细胞之间的亚细胞变化。为了实现这一目标，在这里，我们介绍了一种方法来分析从单个哺乳动物细胞释放的线粒体。骨肉瘤143 B细胞用荧光素特异性10-N-壬基吖啶橙子（NAO）或通过表达荧光蛋白DsRed 2来标记。随后，将单个细胞引入CE-LIF毛细管中，其中通过毛地黄皂苷和胰蛋白酶的组合处理释放细胞器。在这种处理之后，施加电场并且释放的细胞器向LIF检测器电迁移。根据电泳图，计算每个细胞检测到的事件的数量、它们各自的电泳迁移率和它们各自的荧光强度。从DsRed 2标记（保留在完整的线粒体中）和NAO标记（标记所有线粒体）获得的结果是讨论这种单细胞方法的优势和局限性的基础。
Bulk studies are not suitable to describe and study cell-to-cell variation, which is of high importance in biological processes such as embryogenesis, tissue differentiation, and disease. Previously, capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was used to measure the properties of organelles isolated from millions of cells. As such, these bulk measurements reported average properties for the organelles of cell populations. Similar measurements for organelles released from single cells would be highly relevant to describe the subcellular variations among cells. Toward this goal, here we introduce an approach to analyze the mitochondria released from single mammalian cells. Osteosarcoma 143B cells are labeled with either the fluorescent mitochondrion-specific 10-N-nonyl acridine orange (NAO) or via expression of the fluorescent protein DsRed2. Subsequently, a single cell is introduced into the CE-LIF capillary where the organelles are released by a combined treatment of digitonin and trypsin. After this treatment, an electric field is applied and the released organelles electromigrate toward the LIF detector. From an electropherogram, the number of detected events per cell, their individual electrophoretic mobilities, and their individual fluorescence intensities are calculated. The results obtained from DsRed2 labeling, which is retained in intact mitochondria, and NAO labeling, which labels all mitochondria, are the basis for discussion of the strengths and limitations of this single-cell approach.