Stem Cell Identification and Sorting Using the Hoechst 33342 Side Population (SP)

Margaret A. Goodell1

1 Baylor College of Medicine, Houston, Texas
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.18
DOI:  10.1002/0471142956.cy0918s34
Online Posting Date:  November, 2005
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Abstract

This unit describes the use of Hoechst 33342 to identify and purify murine hematopoietic stem cells, the so‐called side population. Three properties of the dye contribute to the ability to distinguish stem cells in this way. Hoechst is a DNA‐binding dye. It has at least two binding modes that result in different spectral properties, allowing resolution of multiple populations by viewing fluorescence at two wavelengths simultaneously. The ability to discriminate SP cells is based on the differential efflux of the dye by a multi‐drug‐like transporter. Particular attention is given to the critical aspects of Hoechst concentration, cell concentration, staining time, and staining temperature.

     
 
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Table of Contents

  • Basic Protocol 1: Staining and Analysis of Stem Cell Side Population in Murine Bone Marrow
  • Support Protocol 1: Preparation of Mouse Bone Marrow
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Staining and Analysis of Stem Cell Side Population in Murine Bone Marrow

  Materials
  • DMEM+ (see recipe)
  • Mouse bone marrow cells prepared according to protocol 2, or bone marrow cells from another species, or cells from non‐bone marrow tissue
  • 1 mg/ml Hoechst 33342 (see recipe)
  • Hanks buffered salt solution+ (HBSS+, see recipe), on ice
  • 200 µg/ml propidium iodide in PBS (see recipe; optional)
  • 5 mM verapamil in 95% ethanol (optional; Sigma)
  • Circulating water bath at exactly 37°C
  • 250‐ml or 50‐ml polypropylene centrifuge tubes (Corning)
  • Flow cytometer with UV laser (usually high‐power argon tuned to 351‐ to 364‐nm excitation) and standard 488‐nm laser
  • 450/20 band‐pass (BP) filter
  • 675‐nm edge filter long‐pass (EFLP; Omega Optical)
  • 610‐nm dichroic mirror short‐pass (DMSP) filter
  • Additional reagents and equipment for counting cells ( appendix 3A)

Support Protocol 1: Preparation of Mouse Bone Marrow

  Materials
  • Mouse (the author uses C57Bl/6, but other strains will work)
  • HBSS+ (see recipe)
  • 70% ethanol in spray bottle
  • Rugged and delicate scissors
  • Small forceps
  • 10‐cm petri dishes
  • 10‐ml syringes
  • 18‐ and 27‐G needles
  • 50‐ml tubes
  • 70‐µm filter or mesh
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Figures

Videos

Literature Cited

Literature Cited
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