Purification of Oligodendrocytes and Their Progenitors Using Immunomagnetic Separation and Percoll Gradient Centrifugation

Raymond J. Colello1, Carmen Sato-Bigbee1

1 Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 3.12
DOI:  10.1002/0471142301.ns0312s03
Online Posting Date:  May, 2001
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Abstract

In this unit, two techniques are described for the purification of oligodendrocytes and their progenitors from the developing mammalian central nervous system (CNS). The first method utilizes the technique of immunomagnetic separation to selectively isolate oligodendrocytes and their progenitor cells from the optic nerve of prenatal and early postnatal rats. This technique takes advantage of the surface antigens expressed on these cells. A paramagnetic bead is attached to the cells via an antibody bridge. Target cells that are coupled to magnetic beads can then be separated from a heterogeneous cell population using a magnetic field. The second method for isolating oligodendrocytes uses Percoll gradient centrifugation to separate oligodendrocytes from a heterogeneous cell population by virtue of their cell density and allows the direct isolation of oligodendrocytes from animals aged postnatal day 4 (P‐4) to adult. This method is particularly useful for assessing physiological systems present in development that may be lost as a result of growing purified neonatal cells in vitro in the absence of neuronal influence.

     
 
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Table of Contents

  • Basic Protocol 1: Purification of Oligodendrocytes and Their Progenitors by Immunomagnetic Separation
  • Basic Protocol 2: Purification of Oligodendrocytes by percoll gradient Centrifugation
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Purification of Oligodendrocytes and Their Progenitors by Immunomagnetic Separation

  Materials
  • 3 M HCl
  • 70%, 85%, 95%, and 100% ethanol
  • 100 µg/ml poly‐L‐lysine (mol. wt. 70,000 to 150,000, Sigma)
  • 0.1% and 5% BSA in PBS, pH 7.4 ( appendix 2A)
  • Rats aged from P‐0 to P‐12
  • CMF‐HBSS ( appendix 2A)
  • 0.05% trypsin/0.5 mM EDTA (e.g., Sigma)
  • 10 µg/ml DNase I/DMEM (see recipe)
  • DMEM/10% FBS (see recipe)
  • Glial cell type–specific antibody
    • For oligodendrocyte progenitors: A2B5 antibody, mouse (IgM) monoclonal  (e.g., Boehringer Mannheim)
    • For oligodendrocytes: O4 antibody (IgM) or anti‐galactocerebroside antibody  (anti‐GalC antibody; IgG), mouse monoclonal (e.g., Boehringer Mannheim)
  • Magnetic beads
    • For oligodendrocyte progenitors: precoated with rat anti‐mouse IgM secondary  antibody (Dynal)
    • For oligodendrocytes: precoated with either goat anti‐mouse IgM or IgG  secondary antibody (Dynal)
  • 4% paraformaldehyde (unit 3.9)
  • PBS, pH 7.4 ( appendix 2A)
  • Fluorescently tagged secondary antibody
  • Blocking solution (see recipe)
  • 5 mg/500 ml 4′,6‐diamidino‐2‐phenylindole (DAPI; Sigma) in recipePBS
  • Aqueous mounting medium (e.g., Aqua poly/Mount, Polysciences)
  • 1% glutaraldehyde/0.1 M cacodylate buffer, pH 7.0
  • Hexamethyldisilazane (HMDS, Ted Pella)
  • 10‐mm glass coverslips
  • 15‐ and 50‐ml conical polypropylene tubes (Falcon)
  • Laminar flow hood, preferably with ultraviolet light source
  • 35‐mm plastic petri dishes, sterile
  • Silicone tubing (i.d. 7 mm, o.d. 9 mm)
  • Pinch clamp
  • Dynal MPC magnet
  • Magnetic particle concentrator (Dynal)
  • Clear tape
  • Dissecting microscope
  • Pasteur pipets, sterile (one flame polished)
  • Dissecting instruments
    • No. 5 forceps, 4 3/4 in. (∼12 cm; e.g., Tiemann)
    • Vannas scissors (e.g., Tiemann)
    • Curved microdissecting scissors, 1/4‐in. (7‐mm) blade (e.g., Tiemann)
    • Scalpel with no. 10 blade
  • Tabletop centrifuge (low speed, 1000 × g)
  • Fluorescence microscope
  • Additional reagents and equipment for dissecting rat optic nerves (unit 3.4)

Basic Protocol 2: Purification of Oligodendrocytes by percoll gradient Centrifugation

  Materials
  • Matrigel (reduced growth factor, Becton Dickinson)
  • Rats aged from P‐4 to adult
  • HEPES/HBSS (see recipe)
  • 10 µg/ml DNase I in HEPES/HBBS (see recipe)
  • Acetyltrypsin solution (see recipe)
  • Isotonic Percoll (see recipe)
  • DMEM/F‐12, pH 7.4 (Life Technologies), containing 1.5% albumin (fatty acid–free, low‐endotoxin bovine serum albumin; Sigma)
  • Chemically defined medium (ODM; see recipe)
  • Dissecting instruments (see protocol 1)
  • Whatman no. 1 filter paper
  • Tabletop centrifuge
  • 15‐ and 50‐ml conical centrifuge tubes
  • 15‐ml Corex tubes
  • High‐speed centrifuge (up to 35,000 × g) with Beckman GA17 rotor
  • Tissue‐treated culture dishes and plates (Corning or Falcon)
  • Cell filtration device: 74‐µm pore size nylon screen (Tetko), ∼10 × 10 cm, and 50‐ to 100‐ml beaker or 70‐µm nylon mesh Falcon cell strainer (Becton Dickinson) on top of a 50‐ml conical tube
  • Additional reagents and equipment for dissecting tissues (unit 3.4)
NOTE: All centrifugations are carried out at 4°C unless otherwise specified.
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Figures

Videos

Literature Cited

Literature Cited
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   Colello, R.J., Wright, A.P., and Fitzgerald, J.J. 1995. Purification of glial cells using immunomagnetic separation. Soc. Neurosci. Abstr. 21:321.
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   Meier, D., Lagenaur, C., and Schachner, M. 1982. Immunoselection of oligodendrocytes by magnetic beads. I. Determination of antibody coupling parameters and cell binding conditions. J. Neurosci. Res. 7:119‐134.
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   Sommer, I. and Schachner, M. 1981. Monoclonal antibodies (O1 and O4) to oligodendrocyte cell surfaces: An immunocytochemical study in the central nervous system. Dev. Biol. 83:311‐327.
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   Wright, A.P., Fitzgerald, J.J., and Colello, R.J. 1997. Rapid purification of glial cells using immunomagnetic separation. J. Neurosci. Methods 74:37‐44.
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Key References
  Berti‐Mattera et al., 1984. See above.
  First description of the use of Percoll gradient centrifugation for the isolation of oligodendrocytes.
  Meier et al., 1982. See above.
  The two papers above are first descriptions of the use of immunomagnetic separation for the isolation of oligodendrocytes.
  Meier and Schachner, 1982. See above.
  First description of the use of immunomagnetic separation for the isolation of oligodendrocyte progenitors and astrocytes.
  Wright et al., 1997. See above.
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