Isolation and characterization of side population cells

Methods Mol Biol. 2005;290:343-52. doi: 10.1385/1-59259-838-2:343.


The protocol for isolation of side population (SP) cells was originally established for murine bone marrow hematopoietic stem cells (HSCs), but it has also been adapted for other species and tissues. This purification strategy offers a simple and reproducible strategy to obtain a highly homogeneous population of HSCs. The method is based on the differential efflux of the fluorescent DNA-binding dye Hoechst 33342 from stem cells relative to nonstem cells. The protocols outlined in this chapter describe the isolation of murine SP cells from both bone marrow and skeletal muscle using the fluorescent DNA-binding dye Hoechst 33342. In these tissues, the SP cells that are isolated are HSCs.

MeSH terms

  • Animals
  • Benzimidazoles / chemistry
  • Bone Marrow Cells / cytology*
  • Cell Separation*
  • Fluorescent Dyes / chemistry
  • Hematopoietic Stem Cells / cytology*
  • Mice
  • Mice, Inbred C57BL


  • Benzimidazoles
  • Fluorescent Dyes
  • bisbenzimide ethoxide trihydrochloride