An improved cell separation technique for marine subsurface sediments: applications for high-throughput analysis using flow cytometry and cell sorting.
An improved cell separation technique for marine subsurface sediments: applications for high-throughput analysis using flow cytometry and cell sorting.
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DOI：
10.1111/1462-2920.12153
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发表时间：
2013-10
影响因子：
5.1
通讯作者：
Inagaki F
中科院分区：
文献类型：
--
作者：
Morono Y;Terada T;Kallmeyer J;Inagaki F
开发了一种从海洋沉积物中分离微生物细胞的改进技术，并对一种高通量且有区分度的细胞计数方法进行了标准化。我们从各种类型的海洋沉积物中分离微生物细胞，然后使用多聚钨酸钠和/或Nycodenz的多层密度梯度回收细胞，结果细胞回收率比以前的方法显著提高。细胞提取效率通常取决于沉积物深度；使用我们开发的新技术，从浅层沉积物样本（深度达100米）中可回收超过80%的总细胞，而从深层样本（深度100 - 365米）中可回收约50%的细胞。分离出的细胞可以使用流式细胞术（FCM）快速计数。在10⁴ - 10⁸个细胞/立方厘米的范围内，数据与手动显微镜直接计数获得的数据非常吻合。我们还证明了可以使用细胞分选仪有效地收集沉积微生物细胞。我们新的细胞分离技术与FCM/细胞分选技术的结合使用，有助于对沉积物中的微生物细胞进行高通量和精确计数，并适用于各种类型的单细胞分析，从而增进我们对很大程度上尚未被描述的深海海底以下生物圈中微生物生命的了解。
Development of an improved technique for separating microbial cells from marine sediments and standardization of a high-throughput and discriminative cell enumeration method were conducted. We separated microbial cells from various types of marine sediment and then recovered the cells using multilayer density gradients of sodium polytungstate and/or Nycodenz, resulting in a notably higher percent recovery of cells than previous methods. The efficiency of cell extraction generally depends on the sediment depth; using the new technique we developed, more than 80% of the total cells were recovered from shallow sediment samples (down to 100 meters in depth), whereas ∼ 50% of cells were recovered from deep samples (100–365 m in depth). The separated cells could be rapidly enumerated using flow cytometry (FCM). The data were in good agreement with those obtained from manual microscopic direct counts over the range 104–108 cells cm−3. We also demonstrated that sedimentary microbial cells can be efficiently collected using a cell sorter. The combined use of our new cell separation and FCM/cell sorting techniques facilitates high-throughput and precise enumeration of microbial cells in sediments and is amenable to various types of single-cell analyses, thereby enhancing our understanding of microbial life in the largely uncharacterized deep subseafloor biosphere.
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影响因子：
56.9
作者：
D'Hondt, S;Jorgensen, BB;Acosta, JLS
通讯作者：
Acosta, JLS
DOI：
10.1073/pnas.1203849109
发表时间：
2012-10-02
影响因子：
11.1
作者：
Kallmeyer, Jens;Pockalny, Robert;D'Hondt, Steven
通讯作者：
D'Hondt, Steven
影响因子：
2.3
作者：
Masui, Noriaki;Morono, Yuki;Inagaki, Fumio
通讯作者：
Inagaki, Fumio
DOI：
10.1073/pnas.1107763108
发表时间：
2011-11-08
影响因子：
11.1
作者：
Morono, Yuki;Terada, Takeshi;Inagaki, Fumio
通讯作者：
Inagaki, Fumio
DOI：
10.1073/pnas.0811793106
发表时间：
2009-07-14
影响因子：
11.1
作者：
D'Hondt, Steven;Spivack, Arthur J.;Stancin, Andrea M.
通讯作者：
Stancin, Andrea M.