Adult mouse hematopoietic stem cells: purification and single-cell assays.
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-/lowc-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.
Ema
Hideo
H
Laboratory of Stem Cell Therapy, Center for Experimental Medicine, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
Morita
Yohei
Y
Yamazaki
Satoshi
S
Matsubara
Azusa
A
Seita
Jun
J
Tadokoro
Yuko
Y
Kondo
Hiroyoshi
H
Takano
Hina
H
Nakauchi
Hiromitsu
H
D000818:Animals; D018952:Antigens, CD34; D002469:Cell Separation / Q000379:methods*; D003114:Colony-Forming Units Assay / Q000379:methods*; D018380:Hematopoietic Stem Cell Transplantation / Q000379:methods*; D006412:Hematopoietic Stem Cells; D051379:Mice; D008810:Mice, Inbred C57BL
D018952:Antigens, CD34