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Flow Cytometry Protocols pp 161–180Cite as

Detection and Enrichment of Hematopoietic Stem Cells by Side Population Phenotype

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Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 263))

Abstract

A flow cytometric procedure has recently been described to isolate hematopoietic stem cells from mouse bone marrow based on the efflux properties of the vital dye Hoechst 33342. The assay defines a subset of cells—termed the “side population” (SP)—by simultaneously measuring fluorescence of the dye at two wavelengths (∼450 nm and >670 nm). In this chapter, SP protocols are provided to detect candidate hematopoietic stem cells in mouse bone marrow and human cord blood. In the standard method, SP profiles are readily observed on a stream-in-air cell sorter using 30 mW of 351–356 nm ultraviolet excitation from a krypton-ion laser. Alternatively, SP profiles can be resolved on an analytical flow cytometer with cuvette flow cell using 8 mW of 325-nm ultraviolet excitation from a helium-cadmium laser. The ability to perform the SP assay on an analytical instrument facilitates optimization of staining conditions to identify hematopoietic and other stem cells in a variety of tissues. It is also demonstrated that SP profiles of slightly lower resolution can be obtained on a stream-in-air cell sorter using 100 mW of 407-nm violet excitation from a krypton-ion laser, raising the possibility that with appropriate validation the SP assay could be performed on flow cytometers that are not equipped with ultraviolet lasers.

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Author information

Authors and Affiliations

  1. Healthspex, Inc., Knoxville, TN

    Shannon S. Eaker

  2. Flow Cytometry Facility and Hematopoiesis Department, Holland Laboratory, American Red Cross, Rockville, MD

    Teresa S. Hawley

  3. Hematopoiesis Department, Holland Laboratory, American Red Cross, Rockville, MD

    Ali Ramezani

  4. Hematopoiesis Department and Blood and Cell Therapy Development, Holland Laboratory, American Red Cross, Rockville, MD

    Robert G. Hawley

  5. Department of Anatomy and Cell Biology, The George Washington University, Washington, DC

    Robert G. Hawley

Authors
  1. Shannon S. Eaker
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  2. Teresa S. Hawley
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  3. Ali Ramezani
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  4. Robert G. Hawley
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Editor information

Editors and Affiliations

  1. American Red Cross, Rockville, MD

    Teresa S. Hawley  & Robert G. Hawley  & 

  2. The George Washington University, Washington, DC

    Robert G. Hawley

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© 2004 Humana Press Inc.

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Eaker, S.S., Hawley, T.S., Ramezani, A., Hawley, R.G. (2004). Detection and Enrichment of Hematopoietic Stem Cells by Side Population Phenotype. In: Hawley, T.S., Hawley, R.G. (eds) Flow Cytometry Protocols. Methods in Molecular Biology™, vol 263. Humana Press. https://doi.org/10.1385/1-59259-773-4:161

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  • DOI: https://doi.org/10.1385/1-59259-773-4:161

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-234-6

  • Online ISBN: 978-1-59259-773-4

  • eBook Packages: Springer Protocols

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