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

Raymond J. Colello¹, Carmen Sato-Bigbee¹

¹Medical 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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Unit Introduction
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
Bibliography
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 PBS
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 Basic 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

Figure 3.12.1

Apparatus used for the magnetic separation of oligodendrocytes and their progenitor cells.

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Figure 3.12.2

Illustration of the Percoll density gradient showing the resultant bands following high-speed centrifugation. Band 1 contains cell debris and myelin, band 2 contains the oligodendrocyte-enriched cell suspension, and band 3 contains red cells.

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Figure 3.12.3

Scanning electron (A, C) and fluorescent micrographs (B, D) showing cultured glial cells that had been immunomagnetically separated (see Basic Protocol 1). The oligodendrocyte progenitors (A, B) and the differentiated oligodendrocytes (C, D) show characteristic morphologies for their stage of development. Note that the beads have little effect on cell outgrowth as demonstrated by the presence of lengthy processes. The fluorescence micrograph (B) shows oligodendrocyte progenitor cells stained with the antibody A2B5 and bound by a FITC-conjugated secondary antibody. The oligodendrocytes (D) were stained with the nuclear stain DAPI.

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Figure 3.12.4

Photomicrographs of cultured oligodendrocytes isolated by Percoll gradient centrifugation (see Basic Protocol 2). Cells were isolated at three different ages and maintained in culture for 2 days (A, B) or 4 days (C). Cells in A and B were isolated from P-5 and P-11 rats, respectively, and were stained with O4 antibody. Cells in C were isolated from P-21 rats and were stained with anti-MBP antibody.

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Literature Cited

Literature Cited
	Abney, E.R., Williams, B.P., and Raff, M.C. 1983. Tracing the development of oligodendrocytes from precursor cells using monoclonal antibodies, fluorescence-activated cell sorting and cell culture. Dev. Biol. 100:166-171.
	Barres, B.A., Hart, I.K., Coles, H.S.R., Burne, J.F., Voyvodic, J.T., Richardson, W.D., and Raff, M.C. 1992. Cell death and control of cell survival in the oligodendrocyte lineage. Cell 70:31-46.
	Barres, B.A., Schmid, R., Sendnter, M., and Raff, M.C. 1993. Multiple extracellular signals are required for long-term oligodendrocyte survival. Development 118:283-295.
	Berti-Mattera, L.N., Larocca, J.N., Pellegrino de Iraldi, A., Pasquini, J.M., and Soto, E.F. 1984. Isolation of oligodendroglial cells from young and adult whole rat brains using an in situ generated Percoll density gradient. Neurochem. Int. 6:41-50.
	Colello, R.J., Wright, A.P., and Fitzgerald, J.J. 1995. Purification of glial cells using immunomagnetic separation. Soc. Neurosci. Abstr. 21:321.
	Collin-Osdoby, P., Ourlser, M.J., Webber, D., and Osdoby, P. 1991. Osteoclast-specific monoclonal antibodies coupled to magnetic beads provide a rapid and efficient method of purifying avian osteoclasts. J. Bone Miner. Res. 6:1353-1365.
	Eisenbarth, G.S., Walsh, F.S., and Nirenburg, M. 1979. Monoclonal antibody to a plasma membrane antigen of neurons. Proc. Natl. Acad. Sci. U.S.A. 76:4913-4917.
	Fok-Seang, J., Mathews, G.A., Ffrench-Constant, C., Trotter, J., and Fawcett, J.W. 1995. Migration of oligodendrocyte precursors on astrocytes and meningeal cells. Dev. Biol. 171:1-15.
	Gard, A.L. and Pfeiffer, S.E. 1990. Two proliferative stages of the oligodendrocyte lineage [A2B5O4- and O4+GalC–] under different mitogenic control. Neuron 5:615-625.
	He, M., Howe, D.G., and McCarthy, K.D. 1996. Oligodendroglial signal transduction systems are regulated by neuronal contact. J. Neurochem. 67:1491-1499.
	Levison, S.W. and McCarthy, K.D. 1992. Astroglia in culture. In Culturing Nerve Cells (G. Banker and K. Goslin, eds.) pp. 309-336. MIT Press, Cambridge, Mass.
	Mage, M.G., McHugh, L.L., and Rothstein, T.L. 1977. Mouse lymphocytes with and without surface immunoglobulin: Preparative scale separation on polystyrene tissue culture dishes coated with specifically purified anti-immunoglobulin. J. Immunol. Methods 15:47-56.
	McCarthy, K.D. and De Vellis, J. 1980. Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J. Cell Biol. 85:890-902.
	Meier, D. and Schachner, M. 1982. Immunoselection of oligodendrocytes by magnetic beads. II. In vitro maintenance of immunoselected oligodendrocytes. J. Neurosci. Res. 7:135-145.
	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.
	Murphy, S.J., Watt, D.J., and Jones, G.E. 1992. An evaluation of cell separation techniques in a model mixed cell population. J. Cell Sci. 102:789-798.
	Rasmussen, A.M., Smeland, E., Erikstein, B.K., Caignault, L., and Funderud, S. 1992. A new method for detachment of Dynabeads from positively selected B lymphocytes. J. Immunol. Methods 146:185-202.
	Richardson, W.D., Pringle, N., Mosley, M.J., Westermark, B., Dubois-Dalcq, M. 1988. A role for platelet-derived growth factor in normal gliogenesis in the central nervous system. Cell 53:309-319.
	Sasaki, A., Levison, S.W., and Ting, J.P.-Y. 1989. Comparison and quantitation of Ia antigen expression on cultured macroglia and amoeboid microglia from Lewis rat cerebral cortex: Analyses and implications. J. Neuroimmunol. 25:63-74.
	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.
	Tuttle, R. and Matthew, W.D. 1991. An in vitro bioassay for neurite growth using cryostat sections of nervous tissue as a substratum. J. Neurosci. Methods 39:193-202.
	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.
	Wysocki, L.J. and Sato, V.L. 1978. “Panning” for lymphocytes: A method for cell selection. Proc. Natl. Acad. Sci. U.S.A. 61:477-483.
	Zeller, N.K., Behar, T.N., Dubois-Dalcq, M.E., and Lazzarini, R.A. 1985. The timely expression of myelin basic protein gene in culturing rat brain oligodendrocytes is independent of continuous neuronal influences. J. Neurosci. 5:2955-2962.
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.
	Meier and Schachner, 1982. See above.
The two papers above are first descriptions of the use of immunomagnetic separation for the isolation of oligodendrocytes.
	Wright et al., 1997. See above.
First description of the use of immunomagnetic separation for the isolation of oligodendrocyte progenitors and astrocytes.