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Isolation and characterization of side population cells
Margaret A Goodell1, Shannon McKinney-Freeman, Fernando D Camargo
1Center for Cell and Gene Therapy, Department of Immunology and Cell and Molecular Biology Program, Baylor College of Medicine, Houston, TX, USA.
Methods in Molecular Biology (Clifton, N.J.)
|September 14, 2004
Summary
Researchers isolated hematopoietic stem cells (HSCs) using the side population (SP) method. This technique utilizes the Hoechst 33342 dye to differentiate stem cells from others in bone marrow and muscle tissue.
Area of Science:
- Stem Cell Biology
- Hematopoiesis
- Cell Sorting
Background:
- The side population (SP) cell isolation protocol was initially developed for murine bone marrow hematopoietic stem cells (HSCs).
- This method has since been adapted for various species and tissues, demonstrating its broad applicability.
- Obtaining a pure population of HSCs is crucial for research and therapeutic applications.
Purpose of the Study:
- To describe the protocol for isolating murine SP cells from bone marrow and skeletal muscle.
- To highlight the utility of the SP method for obtaining a homogeneous population of HSCs.
- To explain the underlying principle of differential dye efflux used in SP cell purification.
Main Methods:
- Utilized the fluorescent DNA-binding dye Hoechst 33342 for cell staining.
- Applied flow cytometry to differentiate SP cells based on dye efflux properties.
- Isolated SP cells from murine bone marrow and skeletal muscle tissues.
Main Results:
- Successfully isolated SP cells from both murine bone marrow and skeletal muscle.
- Demonstrated that the isolated SP cells in these tissues are indeed HSCs.
- Confirmed the reproducibility and simplicity of the SP purification strategy.
Conclusions:
- The side population method provides a reliable strategy for isolating hematopoietic stem cells.
- This technique is effective in multiple murine tissues, including bone marrow and skeletal muscle.
- The differential efflux of Hoechst 33342 dye is the key mechanism for SP cell identification and isolation.