Purifying and Culturing Neural Cells: A Laboratory Manual

by Ben A. Barres, Beth Stevens

Overview

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.

Product Details

ISBN-13:	9781936113996
Publisher:	Cold Spring Harbor Laboratory Press
Publication date:	11/30/2013
Edition description:	Lab Manual
Pages:	205
Product dimensions:	8.50(w) x 10.80(h) x 0.50(d)

Table of 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 im 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