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Isolation and characterization of mouse side population cells
Aysegul V Ergen1, Mira Jeong, Kuanyin K Lin
1The Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX, USA.
Methods in Molecular Biology (Clifton, N.J.)
|November 27, 2012
Summary
Hematopoietic stem cells (HSCs) in mouse bone marrow are found in the side population (SP), identified by their ability to efflux Hoechst dye. This protocol details purifying these highly active SP cells using dye efflux and surface markers.
Area of Science:
- Hematology
- Stem Cell Biology
- Cellular Biology
Background:
- Side population (SP) cells in mouse bone marrow are enriched for hematopoietic stem cell (HSC) activity.
- SP cells are characterized by their ability to efficiently efflux DNA-binding dyes like Hoechst 33342.
- Previous studies confirm SP cells are phenotypically homogeneous and functionally active.
Purpose of the Study:
- To describe a detailed protocol for the purification of murine bone marrow SP cells.
- To leverage Hoechst dye efflux and HSC surface markers for SP cell isolation.
Main Methods:
- Utilizing flow cytometry for cell analysis.
- Employing Hoechst 33342 dye efflux as a primary identification marker.
- Combining dye efflux with the detection of specific HSC surface markers for purification.
Main Results:
- Successful identification of a minor cell subpopulation (SP) within mouse bone marrow.
- Demonstration of efficient Hoechst dye efflux as a hallmark of SP cells.
- Establishment of SP cells as a highly homogeneous and functionally potent stem cell population.
Conclusions:
- The described protocol enables the purification of murine bone marrow SP cells.
- This method combines Hoechst dye efflux with HSC surface marker analysis for effective isolation.
- Purified SP cells represent a valuable resource for studying hematopoietic stem cell biology.
