Adult mouse hematopoietic stem cells: purification and single-cell assays
Adult mouse hematopoietic stem cells: purification and single-cell assays
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DOI：
10.1038/nprot.2006.447
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发表时间：
2006-01-01
期刊：
影响因子：
14.8
通讯作者：
Nakauchi, Hiromitsu
中科院分区：
文献类型：
--
作者：
Ema, Hideo;Morita, Yohei;Nakauchi, Hiromitsu
小鼠造血干细胞 (HSC) 是研究最深入的干细胞，因为小鼠 HSC 的功能测定最早建立，并且小鼠 HSC 的纯化技术进展最快。在这里，我们描述了目前用于纯化 CD34(-/low)c-Kit(+)Sca-1(+)谱系标记(-) (CD34(-)KSL) 细胞的方案，HSC 群体约占成年 C557BL/6 小鼠骨髓细胞的 0.005%。纯化的 HSC 已在细胞和分子水平上进行了表征。由于克隆分析对于 HSC 自我更新和谱系定型的研究至关重要，因此我们在这里介绍我们的单细胞集落测定和单细胞移植程序。我们还介绍了针对少量 HSC 的免疫染色程序，这对于信号转导分析非常有用。 CD34(-)KSL 细胞的纯化需要大约 6 小时。单细胞培养的初始化大约需要 1 小时。单细胞移植大约需要6小时。单细胞免疫染色大约需要 2 d。
Mouse hematopoietic stem cells (HSCs) are the best-studied stem cells because functional assays for mouse HSCs were established earliest and purification techniques for mouse HSCs have progressed furthest. Here we describe our current protocols for the purification of CD34(-/low)c-Kit(+)Sca-1(+)lineage marker(-) (CD34(-)KSL) cells, the HSC population making up approximately 0.005% of bone marrow cells in adult C557BL/6 mice. Purified HSCs have been characterized at cellular and molecular levels. Since clonal analysis is essential for the study of self-renewal and lineage commitment in HSCs, here we present our single-cell colony assay and single-cell transplantation procedures. We also introduce our immunostaining procedures for small numbers of HSCs, which are useful for signal transduction analysis. The purification of CD34(-)KSL cells requires approximately 6 h. Initialization of single-cell culture requires approximately 1 h. Single-cell transplantation requires approximately 6 h. Single-cell immunostaining requires approximately 2 d.