William G Telford1, Ella G Frolova. 1. Experimental Transplantation and Immunology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA. telfordw@mail.nih.gov
Abstract
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. Copyright 2003 Wiley-Liss, Inc.
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. Copyright 2003 Wiley-Liss, Inc.