We (Steve Bartelmez, Ewa Sitnicka, Greg Priestly, and I) have investigated the various emission ranges of Hoechst 33342 in lineage-negative mouse bone marrow cells, and we found differences as Goddel et al. have published. ( Also, see the earlier work of Watson et al. (1985) Cytometry 6: 310.)
In contrast, we found that after gating on viable rhodamine 123-low cells (low means the dimmest 10%), and plotting Hoechst fluorescence (of any color range) versus c-kit (CD 117) expression results in very well defined population of Hoechst-dim, CD117+ cells that has growth and engraftment properties of long-term repopulating stem cells. This phenotype comprises about 0.8% of all lineage negative cells, or about 0.02% of femur bone marrow cells. (Yes, CD34 is not involved in the sorting process, and indeed may not be important in selecting murine stem cells.) Our work was reported at GLIFCA meeting in Detroit last fall.
Dave Coder
dcoder@u.washington.edu
Begin forwarded message:
Date: Thu, 16 Jan 1997 13:59:16 +0000
To: cyto-inbox
From: lssharp@weizmann.weizmann.ac.il (Ayala Sharp)
Subject: Hoechst Red fluorescence?
X-Status:
A paper in J. Exp. Med. Vol. 183 p. 1797 by Goodell et. al.
describes the isolation of Murine Hematopoietic stem cells
using Hoechst 33342 staining. They excite at 350nm and
measure fluorescence at two wavelengths using a 450 BP
(450/20) and 675 EFLP filters with a 610 dichroic mirror.
They show a nice plot of Hoechst Red versus Hoechst Blue.
The literature I've seen shows no Hoechst fluorescence in
the red region. Any comments ? Explanations ? Suggestions ?
Thanks, Ayala.
-------------------------------------------------------------------
Dr. Ayala Sharp
Biological Services,
Weizmann Institute of Science,
Rehovot, ISRAEL.
FAX: 972-8-9344113
E-mail: lssharp@weizmann.weizmann.ac.il
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