Discrimination of the Hoechst side population in mouse bone marrow with violet and near-ultraviolet laser diodes.
BACKGROUND
Discrimination of stem cells with flow cytometric analysis of Hoechst 33342 efflux by the ABCG2 transporter (termed the Hoechst side population, or SP technique) is a valuable methodology for identifying bone marrow progenitors enriched with stem cells. Unfortunately, it requires a ultraviolet (UV) laser source, usually necessitating an expensive and maintenance-intensive argon- or krypton-ion gas laser on a large-scale cell sorter. In this study, we evaluated the ability of recently available violet and near-UV laser diodes to discriminate Hoechst SP on smaller cuvette-based flow cytometers.


METHODS
Violet laser diodes (emitting at 408 and 401 nm) and a near-UV laser diode (emitting at 370 nm) were mounted on a BD Biosciences LSR II and evaluated for their ability to discriminate Hoechst SP in murine bone marrow.


RESULTS
The violet laser diodes discriminated the Hoechst SP, but with poorer resolution than with the standard UV gas laser on a large-scale cell sorter. The near-UV laser diode, in contrast, gave excellent Hoechst SP resolution.


CONCLUSIONS
These evaluations indicated that near-UV laser diodes give excellent Hoechst SP resolution on cuvette-based instruments. As the next generation of cell sorters integrate cuvette-based cell interrogation into conventional jet-in-air cell separation, these laser sources should become applicable for analysis and physical separation of Hoechst SP cells.
Telford
William G
WG
Experimental Transplantation and Immunology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA. telfordw@mail.nih.gov
Frolova
Ella G
EG
D000818:Animals; D001562:Benzimidazoles / Q000378:metabolism; D001854:Bone Marrow Cells / Q000145:classification / Q000166:cytology* / Q000378:metabolism; D002469:Cell Separation / Q000379:methods*; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism; D007834:Lasers; D051379:Mice; D008807:Mice, Inbred BALB C; D010641:Phenotype; D012666:Semiconductors; D014466:Ultraviolet Rays
D001562:Benzimidazoles; D005456:Fluorescent Dyes; C017807:bisbenzimide ethoxide trihydrochloride