Kinetic analyses as a critical parameter in defining the side population (SP) phenotype.
The side population (SP) phenotype has been reported as a method to identify hematopoietic stem cells in the bone marrow based upon differential staining with the fluorescent dye, Hoechst 33342. This technique has drawn great interest in the stem cell community, as it may provide a simple approach to the enrichment of progenitor cells from a variety of normal and malignant tissues. The frequency of these cells and their performance in functional assays has varied considerably within the literature. To investigate mechanisms that may contribute to the SP phenotype, we measured the fluorescence emission of Hoechst-stained bone marrow cells as a function of both time and dye concentration using a custom flow cytometer and data acquisition software. These measurements demonstrate that all nucleated cells within the bone marrow undergo an identical staining pattern at varying rates, even under conditions previously reported to abrogate the SP. Therefore, the SP phenotype is not unique to stem cells, but rather represents a transient feature of marrow cells exposed to Hoechst 33342 for varying amounts of time. We propose that heterogeneity of SP-defined populations may be a consequence of the rate at which differing cell populations accumulate Hoechst 33342. Further, we suggest that dye uptake kinetics will likely be an important factor for optimal use of Hoechst 33342 in isolating stem cells.
Ibrahim
Sherrif F
SF
Institute for Systems Biology, 1441 North 34th Street, Seattle, WA 98103, USA.
Diercks
Alan H
AH
Petersen
Timothy W
TW
van den Engh
Ger
G
D000818:Animals; D001562:Benzimidazoles / Q000378:metabolism / Q000493:pharmacokinetics*; D019070:Cell Lineage / Q000502:physiology*; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism / Q000493:pharmacokinetics; D018380:Hematopoietic Stem Cell Transplantation / Q000379:methods; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000502:physiology; D051379:Mice; D008810:Mice, Inbred C57BL; D010599:Pharmacokinetics; D010641:Phenotype; D012984:Software; D013194:Staining and Labeling / Q000379:methods*; D013997:Time Factors
D001562:Benzimidazoles; D005456:Fluorescent Dyes; C017807:bisbenzimide ethoxide trihydrochloride