title: The use of fluorescent indoline dyes for side population analysis.
abstract: Dye efflux assay evaluated by flow cytometry is useful for stem cell studies. The side population (SP) cells, characterized by the capacity to efflux Hoechst 33342 dye, have been shown to be enriched for hematopoietic stem cells (HSCs) in bone marrow. In addition, SP cells are isolated from various tissues and cell lines, and are also potential candidates for cancer stem cells. However, ultra violet (UV) light, which is not common for every flow cytometer, is required to excite Hoechst 33342. Here we showed that a fluorescent indoline dye ZMB793 can be excited by 488-nm laser, equipped in almost all the modern flow cytometers, and ZMB793-excluding cells showed SP phenotype. HSCs were exclusively enriched in the ZMB793-excluding cells, while ZMB793 was localized in cytosol of bone marrow lineage cells. The efflux of ZMB793 dye was mediated by ATP binding cassette (ABC) transporter Abcg2. Moreover, staining properties were affected by the side-chain structure of the dyes. These data indicate that the fluorescent dye ZMB793 could be used for the SP cell analysis.
authors.affiliation: Department of Biomaterials, Field of Tissue Engineering, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kohara
authors.forename: Kohei
authors.initials: K
authors.lastname: Watanabe
authors.forename: Taichi
authors.initials: T
authors.lastname: Shintou
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Nomoto
authors.forename: Mie
authors.initials: M
authors.lastname: Okano
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Shirai
authors.forename: Takeshi
authors.initials: T
authors.lastname: Miyazaki
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Tabata
mesh_terms: D000818:Animals; D001562:Benzimidazoles / Q000138:chemical synthesis*; D002478:Cells, Cultured; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000138:chemical synthesis*; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D007211:Indoles / Q000138:chemical synthesis*; D051379:Mice; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence / Q000379:methods*
chemical_list: D001562:Benzimidazoles; D005456:Fluorescent Dyes; D007211:Indoles; C057812:indoline; C017807:bisbenzimide ethoxide trihydrochloride