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Purifying and Culturing Neural Cells: A Laboratory Manual


Subject Area(s):  Cell BiologyLaboratory TechniquesNeurobiology

Edited by Ben A. Barres, Stanford University School of Medicine; Beth Stevens, Boston Children's Hospital, Harvard Medical School

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© 2014 • 205 pp., illus. (24 4C, 3 B&W), index
Paperback • $89 62.30
ISBN  978-1-936113-99-6
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Description

Composed of countless neurons, glia, and vascular cells, the nervous system innervates all parts of the body to function as a vast communication network. This complexity makes it challenging to examine neural properties at the cellular and molecular levels. Cell culture systems for specific neural cell types are thus essential for studies of their development and function.

This laboratory manual provides step-by-step protocols for isolating specific cell populations from rodent tissues and culturing them under conditions that closely resemble those in vivo. The contributors describe in detail how to dissect the brain, spinal cord, and other tissues; how to separate cells using mechanical and enzymatic tissue-dissociation strategies; the use of immunopanning and fluorescence-activated cell sorting (FACS) to enrich the target cell population; and the culture conditions that optimize cell viability and growth. Retinal ganglion cells, motor neurons, dorsal root ganglion cells, astrocytes, oligodendrocytes, and Schwann cells are covered, as are vascular cells such as pericytes and endothelial cells. Myelinating cocultures of neurons and oligodendrocytes are also described.

The manual includes detailed recipes for media and reagents, tips for avoiding common pitfalls, and advice for designing new immunopanning protocols using tissues from other sources. Many of the protocols are accompanied by freely accessible online movies that demonstrate critical steps of the procedures. This is an essential laboratory companion for all neurobiologists, from the graduate student level upward.

Contents

Introduction
Ben A. Barres
 
SECTION I. CENTRAL NERVOUS SYSTEM NEURONS
CHAPTER 1
INTRODUCTION
Purification and Culture of Retinal Ganglion Cells
Alissa Winzeler and Jack T. Wang
PROTOCOLS
1 Purification and Culture of Retinal Ganglion Cells from Rodents
Alissa Winzeler and Jack T. Wang
2 Culturing Hybridoma Cell Lines for Monoclonal Antibody Production
Alissa Winzeler and Jack T. Wang
CHAPTER 2
INTRODUCTION
Purification and Culture of Corticospinal Motor Neurons
Wim Mandemakers
PROTOCOLS
1 Retrograde Labeling of Corticospinal Motor Neurons from Early Postnatal Rodents
Wim Mandemakers
2 Immunopanning of Retrograde-Labeled Corticospinal Motor Neurons from Early Postnatal Rodents
Wim Mandemakers
CHAPTER 3
INTRODUCTION
Purification and Culture of Spinal Motor Neurons
David J. Graber and Brent T. Harris
PROTOCOLS
1 Purification and Culture of Spinal Motor Neurons from Rat Embryos
David J. Graber and Brent T. Harris
CHAPTER 4
INTRODUCTION
Purification and Culture of Dorsal Root Ganglion Neurons
J. Bradley Zuchero
PROTOCOLS
1 Purification of Dorsal Root Ganglion Neurons from Rat by Immunopanning
J. Bradley Zuchero
 
SECTION II. ASTROCYTES AND VASCULAR CELLS
CHAPTER 5
INTRODUCTION
Purification and Culture of Astrocytes
Lynette C. Foo
PROTOCOLS
1 Purification of Rat and Mouse Astrocytes by Immunopanning
Lynette C. Foo
2 Purification of Astrocytes from Transgenic Rodents by Fluorescence-Activated Cell Sorting
Lynette C. Foo
CHAPTER 6
INTRODUCTION
Purification and Culture of Central Nervous System Pericytes
Lu Zhou, Fabien Sohet, and Richard Daneman
PROTOCOLS
1 Purification of Pericytes from Rodent Optic Nerve by Immunopanning
Lu Zhou, Fabien Sohet, and Richard Daneman
CHAPTER 7
INTRODUCTION
Purification and Culture of Central Nervous System Endothelial Cells
Lu Zhou, Fabien Sohet, and Richard Daneman
PROTOCOLS
1 Purification of Endothelial Cells from Rodent Brain by Immunopanning
Lu Zhou, Fabien Sohet, and Richard Daneman
 
SECTION III. MYELINATING GLIA
CHAPTER 8
INTRODUCTION
Purification and Culture of Oligodendrocyte Lineage Cells
Jason C. Dugas and Ben Emery
PROTOCOLS
1 Purification of Oligodendrocyte Precursor Cells from Rat Cortices by Immunopanning
Jason C. Dugas and Ben Emery
2 Purification of Oligodendrocyte Lineage Cells from Mouse Cortices by Immunopanning
Ben Emery and Jason C. Dugas
CHAPTER 9
INTRODUCTION
Myelinating Cocultures of Purified Oligodendrocyte Lineage Cells and Retinal Ganglion Cells
Trent A. Watkins and Anja R. Scholze
PROTOCOLS
1 Myelinating Cocultures of Rat Retinal Ganglion Cell Reaggregates and Optic Nerve Oligodendrocyte Precursor Cells
Trent A. Watkins and Anja R. Scholze
CHAPTER 10
INTRODUCTION
Purification of Schwann Cells
Amanda Brosius Lutz
PROTOCOLS
1 Purification of Schwann Cells from the Neonatal and Injured Adult Mouse Peripheral Nerve
Amanda Brosius Lutz
APPENDICES
APPENDIX 1
Designing and Troubleshooting Immunopanning Protocols for Purifying Neural Cells
Ben A. Barres
APPENDIX 2
General Safety and Hazardous Material Information
Index