Series Foreword
Contributors
A User's Guide
Getting Started
Nuts and Bolts
Getting Started
In Defense of Neuronal Cultures
General Principles
Types of Nerve Cell Cultures, Their Advantages and Limitations
History
Primary Cultures Versus Continuous Cell Lines
Primary Cultures
Dissociated-Cell Cultures
Explant or Organotypic Cultures
Reaggregate Cultures
Tumor Cell Lines
Alternative Strategies for Generating Nerve Cell Lines
Glial Cells in Culture
Primary Dissociated Cell Cultures
Source of Tissue
Species
Age
Preparation of Cells
Balanced Salt Solutions and pH Control
Dissecting the Desired Tissue
Dissociation
Purifying Specific Cell Populations
Cell Counting and Assays of Viability
Freezing Cells
Media
Basal Media
Serum
Serum-Free Media
Growth Factors
Depolarization
Antibiotics
Antimitotics
Maintenance of Cultures
Dishes and Substrate
Dishes
Substrate
Untreated Glass or Tissue-Culture Plastic
Polylysine and Polyornithine
Nitrocellulose
Extracellular Matrix Constituents
Other Substrate Molecules
Growth of Neurons on Monolayers of Nonneuronal Cells: Mass Cultures and Microislands
Patterned Substrates, Campenot Chambers, and More
Transfecting Cultured Neurons
Physical Methods
Calcium Phosphate Coprecipitation
Lipid-Mediated Transfection
Microinjection
Bloilstics
Viral Methods
Recombinant HSV
Amplicon-Based HSV
Recombinant Adenovirus
Adeno-Associated Virus
Recombinant Vaccinia Virus
Semliki Forest Virus
Promoters
Concluding Remarks
Acknowledgments
Characterizing and Studying Neuronal Cultures
Bookkeeping
Telling Neurons from Glia
Domain-Specific Markers
Ideatifying Specific Types of Neurons in Culture
Microscopy of Neuronal Cultures
Light Microscopy of Fixed Cultures
Immunostaining
Light-Microscopic Autoradiography
In Site Hybridization
Microscopy of Living Cells
Maintaining Cells on the Microscope Stage
Phototoxicity
Cameras and Recording Devices
Fluorescent Probes
Electron Microscopy of Cultured Neurons
Culture Of Specific Cell Types
Choosing the Right System
Culture and Experimental Use of the PC12 Rat Pheochromocytoma Cell Line
Routine Culture of PC12 Cells
Culture Medium for Cell Growth
Culture Substrate
Collagen
Alternative Coatings For Plastic Dishes
Coatings For Glass
General Maintenance of Cultures
Availability of PC12 Cell Stock Cultures
Preparation and Storage of Frozen PC12 Cells
Generation of Neurite-Bearing PC12 Cell Cultures: Treatment with NGF
Neurite Generation
Neurite Regeneration by Primed PC12 Cells
Washout And removal of NGF
Maintenance in a Defined Medium
Use for Cell Death and Survival Experiments
Counting PC12 Cells
Genetic Manipulation of PC12 Cells
General Approaches
Transfection of PC12 Cells
Genetic Modification of PC12 Cells by Retroviral Infection
Summary and Conclusions
Acknowledgments
Neuronlike Cells Derived in Culture from P19 Embryonal Carcinoma and Embryonic Stem Cells
The P19 System
In Vitro Differentiation of ES Cells into Neuronlike Cells
Advantages and Limitations of the P19 and ES Cell-Based Systems
Protocols
Culture of Stem Cells
Induction of Differentiation
Plating for Neuronal Differentiation
Characterization and Troubleshooting
P19 Cells
ES Cells
Current Uses of the P19 and ES Cell Systems
Culturing the Large Neurons of Aplysia
Protocol for Preparing Cultures of Aplysia Neurons
Single Cells or Dispersed Cells?
Acquisition of Animals
Sterile Technique
Preparation of Culture Medium and of Hemolymph
Preparation of Culture Dishes
Dissection
Removal of Ganglia and Desheathing
Dispersion of Cells in Ganglia
Removal of Individual Neurons
Cars of Cultures
Progression of Cultures
Problems
Other Invertebrate Neurons in Culture
Our Work with Cultured Aplysia Neurons
Note
Culturing Spinal Neurons and Muscle Cells from Xenopus Embryos
Protocol for Obtaining Xenopus Embryos
Obtaining Mature Oocytes
Artificlal Fertilization
Protocol for Early Blastomere Injection
Preparation
Injection
Protecol for Culturing Spinal Neurons and Muscle Cells
Preparation
Microdissection and Cell Plating
Related Techniques and Alternative Methods
Culture Description
Muscle Cells
Spinal Neurons
Other Cell Types
Applications
Neuronal Differentiation and Growth
Synaptegenesis
Transmitter Secretion
Some Future Experiments
Cultures from Chick Peripheral Ganglia
Typos of Peripheral Ganglion Neurons and Their Embryological Origins
Dorsal Root Ganglia
Cranial Sensory Ganglia
Sympathetic Ganglia
Parasympathetic Ganglia
Methods for Culturing Peripheral Ganglion Neurons
Explant Cultures
Dissociated Cell Cultures
Choices of Culture Media
Culture Dishes
Possible Substrates
Our Approach
Protocol
Preparations
Eggs
Coverslips
Sources
Cleaning
Coating with Polyornithine
Coating with Laminin
Culture Medium
Culture Media
Medium for Microscopy
Sylgard Coated Dissection Dish
Dissection and Plating
Setting Up
Dissection
Dissociation and Plating
Separating Neurons from Nonneuronal Cells
Characteristics of the Cultures
Applications
Culturing Mammalian Sympathoadrenal Derivatives
Culture Media and Substrates
L-15 Basal Medium (L-15-Air)
Complete Growth Medium
Serum-Free Growth Medium
Substrates
Culturing Neurons from the Superior Cervical Ganglion
Dissection
Dissociation and Plating
Mechanical Method
Enzymatic Method
Special Culture Formats
Culturing Chromaffin Cells from the Adrenal Medulla
Dissection
Dissociation and Plating
Transdifferentiation of Adrenal Chromaffin Cells
Comments
Acknowledgments
Mass Cultures and Microislands of Neurons from Postnatal Rat Brain
General Culture Protocols
Culture Substrate
Surface Coating
Glial Feeder Layers
Neuronal Growth Medium and Rat Serum
Dissociation and Plating of Neurons
Special Procedures for Microisland Cultures
Preparing Dishes
Preparing Glass Coverslips Coated with Agarose
Preparing Holes in Dishes
Attaching Coverslips with Sylgard
Applying Substrate to Culture Dishes
Alternative Substrates for Microislands
Plating Cells to Make Microisland Cultures
Initial (Glial) Plating
Washing Away Unattached Cellular Material
Second (Neuronal) Plating
Survival and Feeding
Troubleshooting
Properties of Cultured Neurons
Development of Neurons in Miss Cultures
Development of Neurons in Microisland Cultures
Identification of Cultured Neurons
Immunocytochemistry and Visualization of Neurite Patterns in Living Neurons
Acknowledgments
Note
Rat Hippocampal Neurons in Low-Density Culture
The Rationale for Our Approach to Culturing Hippocampal Neurons
Other Approaches for Culturing Hippocampal Neurons
Protocol for Preparing Low-Density Hippocampal Cultures
Preparations
Coverslips
Media
Astrogllal Cell Cultures
Overview
Preparing Primary Glial Cultures
Passaging and Freezing Astroglial Cultures
Maintaining Glial Cultures
Setting out the Neuronal Cultures
Timed Pregnant Rats and Staging of Embryos
Dissection of the Hippocampus
Dissociation and Plating
Feeding and Maintenance of Neuronal Cultures
Characterization of Cultures Prepared by this Method
Troubleshooting
Some All-Too-Familiar Problems
Possible Causes
Substrate
Astroglial Cultures
Media
Applications
Rat Striatal Neurons in Low-Density, Serum-Free Culture
Rationale
Protocol
Tissue Culture Plates
Media
Source of Animals
Dissection of the Striatum
Dissociation and Plating
Maintenance of Striatal Cultures
Appearance and Development of Cultures
What Can Go Wrong?
Culture Substrate
Enzymatic Treatment
Mechanical Dissociation by Trituration
Cell-Plating Density
Applications
Striatal Culture Expression of Functional Neurotrophin Receptors
Neurotrophin Promotion of the Survival of Striatal Neurons in Culture
Specific Neuronal Populations Wherein Survival and Differentiation are limited by the Neurotrophins
Biolegical Responses Indeed by the Neurotrephins
Conclusions
Cell Culture of Cholinergic and Cholinoceptive Neurons from the Medial Habenula
Rationale
Optimal Age for Dissection
Substrate
Dissection Medium
Growth Medium
Growth and Differentiation Factors
Protocol for the Dissection and Culture of MHb Neurons
Preparations
Animals
Substrate
Solutions and Culture Media
Cultures
Dissection of Fetal MHB
Dissociation of Tissue and Culture Conditions
Plating Density
Feeding
Description of Cultures
Troubleshooting
Applications
The Cerebellum: Purification and Coculture of Identified Cell Populations
Development of the Cerebellar Cortex
A Model for Codicil Histogenesis
Cutures of Cerebellar Cells Provide Assays for Specific Steps in Cerebellar Development
Purification of the Cell Classes of the Developing Cerebellar Cortex
The Granule Cell
The Purkinje Cell
Pontine Explants
The Astroglial Cell
General Methodological Strategies
Culture Vessels
Culture Substrates
Culture Media
Source of Cells for Culture
Getting Started
Culture Protocols
Cerebellar Cell Cultures
Dissection
Single-Cell Suspension
Granule Neuron Cultures
Separation of Granule Neurons From Glial and Other Large Cells
Preparation of the Percoll Gradient
Preplating the Granule Cell Fraction
Monolayer Cultures
Reaggregate Cultures
Three-Dimensional Collagen Matrix Cultures
Dye Labeling of Granule Cells
Transplantation of Purified Granule Cells Into Developing Brain
Molecular Studies of Purified Granule Cells
Purkinje Cell Cultures
Growth of R24 Cells for Preparation of GD3 Antibody
Day 1: Coat Dishes
Day 2
Dissection and Dissociation
Separation of Cells by Percoll Gradients
Immunopanning
Plating
Pontine Explant Cultures
Astroglial cultures
Appendix
Recipes
Supplier Index
Acknowledgments
Organotypic Slice Cultures of Neural Tissue
Historical Background
Rationale for Using Organotypic Slice Cultures
Tissue Organization
Accessibility
Cellular Differentiation
Axonal Connectivity
Protocol for Preparing Organotypic Slice Cultures
General Techniques
Culture Medium
Choice of Animals
Dissections
Tissue Slicing
Preparations of Slice Cultures
Roller Tube Cultures
General Procedures
Plasma Clot
Collagen-Coated Coverslips
Preparation of Cultures on Membranes
Care and Maintenance
Characterization of Organotypic Slice Cultures
Hippocampus
Basal Forebrain Cholinergic System
Cerebellum
Troubleshooting
Getting Started
Dissection
Attachment
Why did they die?
Applications
Morphological Experiments
Physiological Experiments
Conclusion
Acknowledgments
Note
Culture of Astrocytes, Oligodendrocytes, and O-2A Progenitor Cells
Astrocytes
The Multifunctional Astrocyte
Astrocyte Purification
Studies on Gilal Progenitor Cells: The O-2A Lineage
The Question of Timing
Studies on O-2A Progenitor Division, Differentiation, and Survival
Precursor Cell Expansion by Cooperating Growth Factors
The O-2Aadult Progenitor
The Oligodendrocyte as a Model System for the Study of Cell Death
N-acetyl-L-cysteine Protection Against Death Induced by TNF-a and by Glutamate
Now NAC and Cillary Neurotrophic Factor Act in Synergy to Protect Oligodendrocytes from Death Induce...
NAC Enhancement of Oligodendrocyte and Neuron Survival in Paradigms of Apoptosis Associated with Exp...
Possible Avenues of Interest in Regard to NAC
Protocol
General Reagents and Methods
Preparing Reagents
Chemically Defined Medium (D-MEM-BS)
Transferrin
Insulin
Enzymes
Trypsin
Collagenase
Soybean Trypsin Inhibitor-Dnase Solution
Ethylenediaminetetraacetic Acid Solution
Preparation of Substrates for Promoting Cell Growth
Preparation of Sterile Coverslips
PLL
Preparation of Growth Factor
Preparation of NAC
Protocol Section II. Immunofluorescence Analysis
Solution Preparation
Hank's Staining Medium
Paraformaldehyde (4%) Solution
Antifade (Dabco)
Indirect Immunofluorescence Analysis
BrdU Labeling of Cells and Anti-BrdU Staining
Protocol Section III, Preparation of Astrocytes and Astrocyte-Conditioned Medium
The Founding Culture
Complement Kill to Remove O-2A Progenitor cells, Type-2 Astrocytes, and Oligodendrocytes
To Make Conditioned Medium
For Pure Astrocyte Monolayers Rather than Conditioned Medium
Protocol Section IV. O-2Aperiastal Progenitor Cells
Mixed Cultures
Purification of O-2A Progenitor Cells
Preparation of Panning Dishes
Preparation Of Cells
Troubleshooting
Growth of O-2A Progenitor Cells and Oligodendrocytes
Expansion of Pure Populations of O-2A Progenitor Cells
Growth of O-2A Progenitors on Astrocyte Monolayers
Pure Cultures of Oligodendrocytes
Pure Cultures of Type-2 Astrocytes
Analysis of Oligodendrocyte Death Induced by Growth Factor WithdrawaI or Exposure to Cytotoxic Agent...
MTT Staining
Death From Cytotoxic Agents
Death from Withdrawal of Trophic Factors
Protocol Section V. Preparation of Primary Cultures of Adult Rat Optic Nerve
Tissue Culture Methods for the Study of Myelination
A Brief History of Myelin Formation in Culture
A New Culture System
Protocols for Preparing Cultures and Inducing Myelination
Housing the Cultures
Substratum Preparation
Collagen Substratum
Poly-L-Lysine-Laminin Substratum
Media Preparation
Maintenance Feeds
Antimitotic Feed
Defined Medium
Myelinating Feeds
Other Media
Dersal Root Ganglion Dissection
Preparation of Pure Neuronal Populations
Preparation of Schwann Cells
The Wood Method
The Brockes Method
Harvesting Perinatal Schwann Cells
Adult-Derived Rat and Human Schwann Cells
Purification
Expansion
Obtaining Myelination by Schwann Cells
Obtaining Myelination by Oligodendrocytes
Histological Options
Electron Microscopy
Sudan Black Staining
Immunocytochemistry
In Situ Hybridization
Autoradiography
Troubleshooting
Infection
Stability of Collagen Substrata
Fibroblast Contamination
Tissue Culture Contribution to Our Understanding of Peripheral Myelin
Acknowledgments
References
Index