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