From: HawleyT@usa.redcross.org
Date: Wed Sep 03 2003 - 12:22:03 EST
Howard wrote -
>A point that remains in contention is whether the violet lasers are
>adequate to define side population (SP) stem cells on the basis of blue and
>red Hoechst 33342 fluorescence. BD seems to think so; others don't. I am
>hoping Bill Telford and/or Bob Hawley will weigh in on this subject in
>response to your posting.
Bob asked me to post his response... Teresa.
Hi everyone,
I cannot provide any information about whether it is possible to use a violet diode to
perform the side population (SP) assay to detect candidate hematopoietic stem cells
(HSCs). Until we receive the violet diode laser for our BD LSR, I can only speculate as
to whether it might be feasible on our modified machine (we have been successful using 8
mW of UV excitation from the HeCd laser on this platform). At the moment, Bill Telford's
experience with the violet diode on his LSRII indicates that it cannot be
readily implemented, even with the enhanced collection optics on the newer instrument.
On the other hand, as Marty Bigos pointed out in an earlier posting (June 16, 2003), he
could carry out the SP assay using Hoechst 33342 excited by 200 mW of 407 nm from a
krypton laser. In our hands, 100 mW of 407 nm from a krypton laser was capable of
generating an SP profile on the FACSVantage SE/DiVa using mouse bone marrow cells (albeit
at lower resolution than with UV excitation). We confirmed that the majority of cells
falling within the SP region co-expressed c-kit and Sca-1, consistent with the murine
HSC phenotype (attached Fig. 3; preprint of the article available upon request), and that
the SP profile was dependent on active efflux of the dye since it could be reduced by
fumitremorgin C, a specific inhibitor of the ABCG2 transporter (attached Fig. 6).
We tried once (Teresa, Bill et al.) to generate an SP profile with Hoechst 34580 using
407 nm excitation from the krypton laser but obtained negative results. We decided not to
pursue it further. Even if an SP profile is observed, the identity of cells falling in
the SP region will need to be stringently validated. Given the inherent variability of
the SP assay, all things considered, UV excitation remains our choice at the present
time.
Best regards,
Bob Hawley
-------------------------------
Robert G. Hawley, Ph.D.
Executive Director, Cell Therapy Research and Development
and Head, Hematopoiesis Department
Holland Laboratory, American Red Cross
15601 Crabbs Branch Way
Rockville, MD 20855
Professor of Anatomy and Cell Biology
Programs in Genetics and Molecular and Cellular Oncology
The George Washington University
Washington, DC
Editor-in-Chief
Current Gene Therapy
Tel: 301-738-0420
Fax: 301-738-0444
E-mail: hawleyr@usa.redcross.org
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