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First published online December 21, 2006
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2006-0567v1
25/4/1029    most recent
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Submitted on September 7, 2006
Accepted on December 13, 2006

Technology Development

Side Population Analysis Using a Violet-Excited Cell Permeable DNA Binding Dye

William G. Telford 1*, Jolene Bradford 2, William Godfrey 2, Robert W. Robey 3, Susan E. Bates 3

1 Experimental Transplantation and Immunology Branch, NCI-NIH, Bethesda, Maryland
2 Molecular Probes Invitrogen, Eugene, Oregon
3 Cancer Therapeutics Branch, NCI-NIH, Bethesda, Maryland

* To whom correspondence should be addressed. E-mail: telfordw{at}mail.nih.gov.


  Abstract

Hoechst 33342 side population (SP) analysis is a common method for identifying stem cells in mammalian hematopoetic and non-hematopoetic tissues. While widely employed for stem cell analysis, this method requires an ultraviolet laser to excite Hoechst 33342. Flow cytometers equipped with UV sources are not common due to the cost of both the laser and optics that can transmit light UV light. Violet laser sources are inexpensive and are now common fixtures on flow cytometers, but have been previously shown to provide insufficient Hoechst dye excitation for consistent resolution of SP cells.

One solution to this problem is to identify additional fluorescent substrates with the same pump specificity as Hoechst 33342, but with better violet excitation characteristics. DyeCycleTM Violet reagent has emission characteristics similar to Hoechst 33342, but with a longer wavelength excitation maxima (369 nm). When this dye is loaded into hematopoetic cells, a sharply resolved "side population" was also observed, similar in appearance to that seen with Hoechst 33342. Unlike Hoechst SP, DCV SP was similar in appearance with both violet and UV excitation. DCV SP could be inhibited fumetrimorgin C, and showed the same membrane pump specificity as Hoechst 33342. Simultaneous immunophenotyping with stem cell markers in mouse bone marrow demonstrated that DCV SP was restricted to the stem cell LSK population, as is Hoechst SP. Pending confirmation by functional analysis of DCV SP cells, these results suggest that DCV efflux identified approximately the same stem cell population as Hoechst 33342 efflux. Substituting DCV for Hoechst 33342 in the SP technique may therefore allow side population analysis on flow cytometers with violet lasers.

Key Words. stem cell, Hoechst 33342, side population, DyeCycle, bone marrow, cord blood






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