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Isolation and Purification of Primary Rodent Astrocytes

David E. Weinstein1

1Albert Einstein College of Medicine, Bronx, NewYork

Publication Name: 
Current Protocols in Neuroscience
Unit Number: 
UNIT 3.5
DOI: 
10.1002/0471142301.ns0305s00
Online Posting Date: 
May, 2001
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Abstract

Astrocytes are a major cell type in the mammalian central nervous system (CNS). The ability to obtain virtually pure populations of these cells makes it possible to study their function as isolated cells or in mixed populations where they support the growth and survival of surrounding neurons. Unlike other mature CNS cells, mature astrocytes maintain the lifelong ability to reenter the cell cycle. The first isolation procedure described in this unit takes advantage of the proliferative ability of these cells, as does the second, except that no antibody or complement treatment is required. A procedure for detecting glial fibrillary acidic protein (GFAP), which is present in most astrocytes in vivo and virtually all astrocytes in vitro and is a useful marker for assessing the purity of cultures, is also presented.

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol: Isolation and Culture of Astrocytes from Rodent Cerebellum
  • Alternate Protocol: Enrichment of Astrocytes by Differential Adhesion
  • Support Protocol: Immunohistochemical Detection of Glial Fibrillary Acidic Protein (GFAP)
  • Reagents and Solutions
  • Commentary
  • Bibliography
  • Figures
     
 
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Materials

Basic Protocol: Isolation and Culture of Astrocytes from Rodent Cerebellum

 Materials
  • Mice aged P-0 to P-7
  • PBS/glucose (see recipe)
  • 60% and 30% Percoll (see recipes)
  • Trypsin/DNase solution (see recipe)
  • DNase solution (see recipe)
  • PBS/glucose (see recipe) containing 5 mg/liter MgSO4
  • DMEM/10% FBS (appendix 2A) containing 2% glucose
  • 0.4% trypan blue solution (e.g., Life Technologies)
  • Tissue culture plates coated with poly-l- (or poly-d-) lysine (appendix 2A)
  • PBS (appendix 2A)
  • Horse serum (optional)
  • 0.05% trypsin/0.53 mM EDTA (Life Technologies)
  • Anti–Thy 1.1 or 1.2 antibody; depending on the species and strain of animals used as the source of astrocytes (if monoclonal, IgM antibodies are preferable)
  • Dulbecco's minimum essential medium (DMEM)
  • Rabbit or guinea pig complement (Life Technologies)
  • Fetal bovine serum (FBS, appendix 2A; optional)
  • Dimethyl sulfoxide (DMSO; optional)
  • Dissecting instruments:
    • 1 pair microdissecting scissors (~3.5-in.)
    • 1 pair dissecting scissors (~6.5-in)
    • 2 pairs no. 5 Dumont forceps (~4.75-in.)
    • 1 pair Semkin dissecting forceps (~6-in)
    • 1 pair curved no. 5 Dumont forceps (optional)
  • 100-mm petri dishes (Falcon)
  • 5-ml and 15-ml conical tubes (Falcon)
  • 9-in. Pasteur pipets (one flame polished) and bulb
  • Swinnex 13-mm filter units (Millipore) with 20-µm nylon mesh filters (Fisher)
  • 100-mm tissue culture plates (Falcon)
  • 1-ml syringes
  • 18-, 20-, and 23-G needles
  • Humidified 37°C, 7% CO2 incubator
  • Bright-field microscope equipped for epifluoresence
  • Additional reagents and equipment for determining cell number and viability with a hemacytometer and trypan blue, and trypsinizing cells (See cpmb appendix 3F and appendix 1A in this manual)

NOTE: The following procedures must be carried out under sterile conditions on a clean bench or in a laminar flow hood.

NOTE: All protocols using live animals must be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals.


Alternate Protocol: Enrichment of Astrocytes by Differential Adhesion

 Additional Materials (also see Basic Protocol)
  • PBS (appendix 2A), 37°C

Support Protocol: Immunohistochemical Detection of Glial Fibrillary Acidic Protein (GFAP)

 Materials
  • Astrocyte culture (see Basic Protocol or Alternate Protocol)
  • Glass coverslips or 8-well slides (Lab-Tek, Nalge Nunc) coated with poly-l- (or poly-d-) lysine (appendix 2A)
  • PBS (appendix 2A)
  • 100% methanol, ice-cold
  • Blocking reagent: 10% goat serum/0.1% Triton X-100 in PBS
  • Anti-GFAP monoclonal antibody (clone GA-5; Boehringer Mannheim)
  • Anti-mouse antiserum conjugated with chromofluor or enzyme
Looking for materials?  Suppliers Guide
     
 
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Figures

  • Figure 3.5.1
    Neonatal rodent cerebellum. The arrowheads indicate the rostral and caudal borders of the developing cerebellum. Harvesting of this tissue is accomplished by passing Dumont forceps under the cerebellum at the sites of the lateral arrowheads and gently lifting the tissue up, separating the cerebellum from the mid- and hindbrain.

    View Image
  • Figure 3.5.2
    Discontinuous Percoll gradients. Enrichment of astrocytes from a heterogeneous cerebellar cell suspension is achieved by passing the cells over a Percoll gradient. The small, dense granule cell neurons pass through the 30% Percoll and come to rest on a cushion of 60% Percoll. The larger, membranous astrocytes stop at the medium/30% Percoll interface. The 60% Percoll will be blue, the 30% Percoll will be white, and the loading medium will be red.

    View Image

Literature Cited

 Literature Cited
    Dobrenis, K., Makman, M.H., and Stefano, G.B. 1995. Occurrence of the opiate alkaloid-selective mu3 receptor in mammalian microglia, astrocytes and Kupffer cells. Brain Res. 686:239-248.
    Fujita, S. 1967. Quantitative analysis of cell proliferation and differentiation in the cortex of the postnatal mouse cerebellum. J. Cell Biol. 32:277-287.
    Hatten, M.E. 1985. Neuronal regulation of astroglial morphology and proliferation in vitro. J. Cell Biol. 100:384-396.
    Hatten, M.E. 1987. Neuronal inhibition of astroglial cell proliferation is membrane mediated. J. Cell Biol. 104:1353-1360.
    Hatten, M.E., Lynch, M., Rydel, R.E., Sanchez, J., Joseph-Silverstein, J., Moscatelli, D., and Rifkin, D.B. 1988. In vitro neurite extension by granule neurons is dependent upon astroglial-derived fibroblast growth factor. Dev. Biol. 125:280-289.
    Latov, N., Nilaver, G., Zimmerman, E.A., Johnson, W.G., Silverman, A.J., Defendini, R., and Cote, L. 1979. Fibrillary astrocytes proliferate in response to brain injury: A study combining immunoperoxidase technique for glial fibrillary acidic protein and radioautography of tritiated thymidine. Dev. Biol. 72:381-384.
    Luskin, M.B., Pearlman, A.L., and Sanes, J.R. 1988. Cell lineage in the cerebral cortex of the mouse studied in vivo and in vitro with a recombinant retrovirus. Neuron 1:635-647.
    Price, J. and Thurlow, L. 1988. Cell lineage in the rat cerebral cortex: A study using retroviral-mediated gene transfer. Development 104:473-482.
    Price, J., Turner, D., and Cepko, C. 1987. Lineage analysis in the vertebrate nervous system by retrovirus-mediated gene transfer. Proc. Natl. Acad. Sci. U.S.A. 84:156-160.
    Sturrock, R.R. 1975. A quantitative electron microscopic study of myelination in the anterior limb of the anterior commissure of the mouse brain. J. Anat. 119:67-75.
    Swanson, R.A., Liu, J., Miller, J.W., Rothstein, J.D., Farrell, K., Stein, B.A., and Longuemare, M.C. 1997. Neuronal regulation of glutamate transporter subtype expression in astrocytes. J. Neurosci. 17:932-940.
    Walsh, C. and Cepko, C.L. 1988. Clonally related cortical cells show several migration patterns. Science 241:1342-1345.
    Weinstein, D.E., Shelanski, M.L., and Liem, R.K. 1990. C17, a retrovirally immortalized neuronal cell line, inhibits the proliferation of astrocytes and astrocytoma cells by a contact-mediated mechanism. Glia 3:130-139.
    Weinstein, D.E., Shelanski, M.L., and Liem, R.K. 1991. Suppression by antisense mRNA demonstrates a requirement for the glial fibrillary acidic protein in the formation of stable astrocytic processes in response to neurons. J. Cell Biol. 112:1205-1213.
    Wetts, R. and Fraser, S.E. 1988. Multipotent precursors can give rise to all major cell types of the frog retina. Science 239:1142-1145.
 Key References
    Hatten, 1985. See above.

First description of the use of a discontinuous gradient for separation of astrocytes and neurons.

    Weinstein, et al., 1990. See above.

Describes in detail the analysis of astrocyte response to a neuronal cell line.

     
 
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