Single-Cell Isolation and Cloning of Tetrahymena thermophila Cells with a Fluorescence-Activated Cell Sorter
Single-Cell Isolation and Cloning of Tetrahymena thermophila Cells with a Fluorescence-Activated Cell Sorter
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DOI：
10.1111/j.1550-7408.2010.00517.x
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发表时间：
2011-01-01
影响因子：
2.2
通讯作者：
Yomo, Tetsuya
中科院分区：
文献类型：
--
作者：
Mori, Kotaro;Kashiwagi, Akiko;Yomo, Tetsuya
我们开发了一种在化学成分确定的培养基（CDM）中使用荧光激活细胞分选仪（FACS）克隆纤毛虫嗜热四膜虫细胞的方法。虽然T.嗜热菌是一种典型的单细胞真核生物，其克隆存在两大技术难题。首先，T。嗜热菌不能从CDM中的低密度增殖，特别是如果接种物含有单细胞。第二，一般的克隆方法是耗时的并且具有低通量。在这里，我们通过添加牛血清白蛋白来修改CDM，所述牛血清白蛋白有助于从密度为10个细胞/ml（1个细胞/100 μ l）的接种物生长。此外，我们应用流式细胞仪分离单细胞。我们表明，有可能根据吞噬的荧光珠的存在或不存在来分离细胞群，并通过FACS在改良的CDM中分离单细胞。我们的技术允许直接分离单细胞并促进克隆菌株的建立。
We developed a method for cloning cells of the ciliate Tetrahymena thermophila in chemically defined medium (CDM) using a fluorescence-activated cell sorter (FACS). Although T. thermophila is a model unicellular eukaryote, two major technical difficulties remain in its cloning. First, T. thermophila fails to proliferate from low density in CDM, particularly if the inoculum contains single cells. Second, general cloning methods are time consuming and have low throughput. Here, we modified the CDM by addition of bovine serum albumin that helped growth from an inoculum with a density of 10 cell/ml (1 cell/100 mu l). In addition, we applied a FACS for isolation of single cells. We showed that it is possible to separate cell populations based on the presence or absence of phagocytosed fluorescent beads and to isolate single cells in a modified CDM by FACS. Our techniques allow the direct isolation of single cells and facilitate the establishment of clonal strains.