Isolation and Generation of Oligodendrocytes by Immunopanning

Margot Mayer‐Pröschel1

1 Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 3.13
DOI:  10.1002/0471142301.ns0313s00
Online Posting Date:  May, 2001
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Abstract

This unit presents a procedure for the purification of oligodendrocyte progenitor cells, their expansion in vitro, and differentiation of these cells to yield oligodendrocyte cultures. A variation is also presented, detailing the direct isolation of differentiated oligodendrocytes from postnatal brain. The purification of the target cell population is achieved by exploiting the differential binding of cells to tissue culture dishes coated with an antibody directed against a specific cell‐surface antigen. Cells expressing this surface antigen are retained on the dish and are thereby separated from the remaining cell population.

     
 
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Table of Contents

  • Basic Protocol 1: In Vitro Generation of Pure Oligodendrocyte Cultures
  • Support Protocol 1: Dissection of Corpus Callosum from Rat Pups
  • Alternate Protocol 1: Direct Isolation of Oligodendrocytes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: In Vitro Generation of Pure Oligodendrocyte Cultures

  Materials
  • Anti‐IgG (H + L) antibody or anti‐IgM antibody (Southern Biotechnology)
  • recipe50 mM Tris⋅Cl, pH 9.5 ( appendix 2A)
  • Cell‐type‐specific antibody solutions (see recipe): anti‐Ran‐2 antibody and anti‐GalC antibody (for negative selection) and anti‐A2B5 antibody or anti‐O4 antibody (for positive selection)
  • DMEM medium (e.g., Life Technologies), serum‐free
  • Ten to twenty 7‐day‐old rat pups
  • 1.3% collagenase solution (see recipe)
  • 30 U/ml papain solution, activated (see recipe)
  • Inhibitor solution (see recipe)
  • DMEM‐BS medium (see recipe)
  • DMEM‐BS (see recipe) containing 10 ng/ml platelet‐derived growth factor (PDGF; see recipe for growth factors) and 10 ng/ml basic fibroblast growth factor (bFGF; see recipe for growth factors)
  • DMEM‐BS (see recipe) with thyroid hormone omitted from Sato mix and containing 10 ng/ml PDGF, 5 ng/ml neurotrophin‐3 (NT‐3) and 20 ng/ml forskolin (see recipe for growth factors)
  • DMEM‐BS (see recipe) containing 40 ng/ml insulin‐like growth factor I (IGF‐I; see recipe for growth factors)
  • 100‐mm Nunclon tissue culture dishes (Nunc)
  • Dissecting instruments
  • 19‐, 23‐, and 27‐G needles
  • 1‐ml plastic disposable syringes
  • Cell scraper (Costar), sterile
  • recipePoly‐L‐lysine–coated tissue culture dishes or coverslips ( appendix 3044)
  • Additional reagents and equipment for tissue culture (see CPMB APPENDIX and appendix 1A in this manual) and dissection of corpus callosum (see protocol 2) or optic nerve (unit 3.4)
NOTE: All culture incubations are performed in a humidified 37°C, 7.5% CO 2 incubator unless otherwise specified.NOTE: All reagents and equipment coming into contact with live cells must be sterile, and proper sterile technique should be followed accordingly.
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Figures

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Literature Cited

Literature Cited
   Barnett, S.C., Hutchins, A.‐M., and Noble, M. 1993. Purification of olfactory nerve ensheathing cells from the olfactory bulb. Dev. Biol. 155:337‐350.
   Barres, B.A., Schmidt, R., Sendtner, M., and Raff, M.C. 1993. Multiple extracellular signals are required for long‐term oligodendrocyte survival. Development 118:283‐295.
   Bartlett, P.F., Noble, M.D., Pruss, R.M., Raff, M.C., Rattray, S., and Williams, C.A. 1980. Rat neural antigen‐2 (Ran‐2): A cell surface antigen on astrocytes, ependymal cells, Muller cells and leptomeningeal cells. Brain Res. 204:339‐351.
   Bögler, O. and Noble, M. 1994. Measurement of time in oligodendrocyte‐type‐2 astrocyte (O‐2A) progenitors is a cellular process distinct from differentiation or division. Dev. Biol. 162:525‐538.
   Bögler, O., Wren, D., Barnett, S.C., Land, H., and Noble, M. 1990. Cooperation between two growth factors promotes extended self‐renewal, and inhibits differentiation, of O‐2A progenitor cells. Proc. Natl. Acad. Sci. U.S.A. 87:6368‐6372.
   Bottenstein, J.H. and Sato, G.H. 1979. Growth of a rat neuroblastoma cell line in serum‐free supplemented medium. Proc. Natl. Acad. Sci. U.S.A. 76:514‐517.
   Eisenbarth, G.S., Walsh, F.S., and Nirenberg, M. 1979. Monoclonal antibodies to a plasma membrane antigen of neurons. Proc. Natl. Acad. Sci. U.S.A. 76:4913‐4916.
   Groves, A.K., Barnett, S.C., Franklin, R.J.M., Crang, A.J., Mayer, M., Blakemore, W.F., and Noble, M. 1993. Repair of demyelinated lesions by transplantation of purified O‐2A progenitor cells. Nature 362:453‐455.
   Ibarolla, N., Mayer, M., Rodriguez‐Pena, A., and Noble, M. 1996. Evidence for the existence of two timing mechanisms in oligodendrocyte development. Dev. Biol. 180:1‐21.
   Mayer, M. and Noble, M. 1994. N‐Acetyl‐L‐cysteine is a pluripotent protector against cell death and enhancer of trophic‐factor mediated cell survival in vitro. Proc. Natl. Acad. Sci. U.S.A. 91:7496‐7500.
   Mayer, M., Bhakoo, K., and Noble, M. 1994. Ciliary neurotrophic factor and leukemia inhibitory factor promote the generation, survival and maturation of oligodendrocytes in vitro. Development 120:143‐153.
   Ranscht, B., Clapshaw, P.A., Price, J., Noble, M., and Seifert, W. 1982. Development of oligodendrocytes and Schwann cells studied with a monoclonal antibody against galactocerebroside. Proc. Natl. Acad. Sci. U.S.A. 79:2709‐2713.
   Sommer, I. and Schachner, M. 1981. Monoclonal antibodies (O1 and O4) to oligodendrocyte cell surfaces: An immunological study in the central nervous system. Dev. Biol. 83:311‐327.
   Wysocki, L.J. and Sato, V.L. 1978. “Panning” for lymphocytes: A method for cell selection. Proc. Natl. Acad. Sci. U.S.A. 75:2844‐2848.
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