Flow Cytometric Sorting of Neuronal and Glial Nuclei From Central Nervous System Tissue
Flow Cytometric Sorting of Neuronal and Glial Nuclei From Central Nervous System Tissue
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DOI：
10.1002/jcp.22365
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发表时间：
2011-02-01
影响因子：
5.6
通讯作者：
Ohkawa, Yasuyuki
中科院分区：
文献类型：
--
作者：
Okada, Seiji;Saiwai, Hirokazu;Ohkawa, Yasuyuki
由于中枢神经系统（CNS）复杂的细胞异质性，获得具有细胞类型特异性的分子描述相对困难。特别是，表观遗传调控或分子谱的比较分析受到缺乏足够的方法从CNS组织中选择性纯化确定的细胞群的阻碍。在这里，我们开发了一种直接纯化策略的神经细胞核从中枢神经系统组织的荧光激活细胞分选（FACS）的基础上。我们成功地从复杂组织如脑、脊髓、肝、肾和骨骼肌中分离出细胞核，这些组织通过机械或化学方法挤出，分离出的细胞核结构保持不变，并含有核蛋白和核DNA/RNA。我们收集了足够数量的细胞核，从神经元和少突胶质细胞使用免疫标记的核蛋白的流式细胞仪或从遗传标记的转基因小鼠。此外，从木瓜蛋白酶抗体库中分离的Fab片段的使用，有效地丰富了专门的细胞群，显着提高免疫标记的功效。这种方法可以应用于各种各样的异质性组织，是至关重要的了解细胞特异性的信息染色质动力学，核蛋白，蛋白质-DNA/RNA相互作用，并保留在细胞核中的转录组，如非编码RNA。J.细胞。226：552-558，2011。(C)2010 Wiley-Liss，Inc.
Due to the complex cellular heterogeneity of the central nervous system (CNS), it is relatively difficult to reliably obtain molecular descriptions with cell-type specificity. In particular, comparative analysis of epigenetic regulation or molecular profiles is hampered by the lack of adequate methodology for selective purification of defined cell populations from CNS tissue. Here, we developed a direct purification strategy of neural nuclei from CNS tissue based on fluorescence-activated cell sorting (FACS). We successfully fractionated nuclei from complex tissues such as brain, spinal cord, liver, kidney, and skeletal muscle extruded mechanically or chemically, and fractionated nuclei were structurally maintained and contained nucleoproteins and nuclear DNA/RNA. We collected sufficient numbers of nuclei from neurons and oligodendrocytes using FACS with immunolabeling for nucleoproteins or from genetically labeled transgenic mice. In addition, the use of Fab fragments isolated from papain antibody digests, which effectively enriched the specialized cell populations, significantly enhanced the immunolabeling efficacy. This methodology can be applied to a wide variety of heterogeneous tissues and is crucial for understanding the cell-specific information about chromatin dynamics, nucleoproteins, protein-DNA/RNA interactions, and transcriptomes retained in the nucleus, such as non-coding RNAs. J. Cell. Physiol. 226: 552-558, 2011. (C) 2010 Wiley-Liss, Inc.