Adult mouse hematopoietic stem cells: purification and single-cell assays

被引:142
作者
Ema, Hideo
Morita, Yohei
Yamazaki, Satoshi
Matsubara, Azusa
Seita, Jun
Tadokoro, Yuko
Kondo, Hiroyoshi
Takano, Hina
Nakauchi, Hiromitsu
机构
[1] Univ Tokyo, Inst Med Sci, Ctr Med Expt, Lab Stem Cell Therapy,Minato Ku, Tokyo 1088639, Japan
[2] ReproCell Inc, Chiyoda Ku, Tokyo 1000011, Japan
[3] Musashino Res Cross Hosp, Dept Hematol & Oncol, Tokyo 1808610, Japan
关键词
D O I
10.1038/nprot.2006.447
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:2979 / 2987
页数:9
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