NEURONAL CELL CULTURE : methods and protocols.

Preface
Acknowledgments
Contents
Contributors
Chapter 1: General Overview of Neuronal Cell Culture
	References
Chapter 2: Considerations for the Use of SH-SY5Y Neuroblastoma Cells in Neurobiology
	1 Introduction
		1.1 Undifferentiated Versus Differentiated SH-SY5Y Cells
		1.2 Retinoic  Acid
		1.3 Phorbol Esters
		1.4 Dibutyryl Cyclic  AMP
		1.5 Additional Methods of Differentiation
		1.6 Markers for Differentiation
		1.7 Markers for Receptors/Transporters
			1.7.1 Dopaminergic Neurons
		1.8 Adrenergic Neurons
		1.9 Cholinergic Neurons
	2 Materials
		2.1 Cells
		2.2 Reagents
	3 Methods
		3.1 Initial Culture Conditions
		3.2 Subculture Conditions
		3.3 Differentiation
		3.4 Replating After Differentiation
		3.5 Tips for Confocal Imaging with SHSY Cells
		3.6 Freezing Down
	4 Notes
	References
Chapter 3: Cultured Cell Line Models of Neuronal Differentiation: NT2, PC12, and SK-N-MC
	1 Introduction
	2 Materials
	3 Methods
		3.1 Cell Culture
		3.2 Treatment
		3.3 Transfection
		3.4 Protein Transduction and Fluorescence Staining
		3.5 Conclusion
	4 Notes
	References
Chapter 4: Murine Teratocarcinoma-Derived Neuronal Cultures
	1 Introduction
	2 Materials and Equipment
		2.1 Cell Lines
		2.2 Growth and Differentiation Media
			2.2.1 Freeze Media
			2.2.2 Preparation of Retinoic  Acid
			2.2.3 Preparation of Dibutyryl-cAMP (db-cAMP)
	3 Methods
		3.1 Maintenance and Propagation of F9 Cells in Culture
		3.2 Maintenance and Propagation of P19 Cells in Culture
		3.3 Freezing Cells
		3.4 Induction of Neuronal Differentiation of F9 Cells
		3.5 Induction of Neuronal Differentiation of P19 Cells
		3.6 Protocol for Generation of Serotonergic or Catecholaminergic Neurons from F9 Cells
			3.6.1 Protocol for Generation of High-Yield Enriched Neuronal Cultures from P19 Cells
	4 Notes
	References
Chapter 5: Isolation and Propagation of Primary Human and Rodent Embryonic Neural Progenitor Cells and Cortical Neurons
	1 Introduction
	2 Materials
		2.1 Brain Tissue
		2.2 Reagents
		2.3 Equipment
	3 Methods
		3.1 Initiation (Fig. 1)
		3.2 Isolation and Expansion of Primary Human Embryonic Neural Progenitor Cells (hNPC) (Fig. 2)
		3.3 Differentiation of hNSC into the Oligodendrocyte Lineage (Fig. 3)
		3.4 Characterization of NPC and  OPC
			3.4.1 Immunostaining
			3.4.2 RT-QPCR
		3.5 Isolation and Maintenance of Primary Human Embryonic Cortical Neurons (Fig. 4)
		3.6 Analysis of the Expression of Genes and Proteins Involved in Synapse Function Using Human Synaptic Plasticity PCR Array
	4 Notes
	References
Chapter 6: Mouse Enteric Neuronal Cell Culture
	1 Introduction
	2 Materials
		2.1 Isolation of Smooth Muscle/Myenteric Plexus (SM/MP) Strips
		2.2 Reagents
	3 Methods
		3.1 SM/MP Strips from Mouse  Gut
		3.2 Enzymatic Dissociation of SM/MP Tissues
		3.3 Culture of Primary Enteric Neurons
		3.4 Culture and Differentiation of Enteric Neural Stem/Progenitor Cells (See Note 7)
			3.4.1 Primary Neurosphere Culture of Enteric Neural Stem/Progenitor Cells
			3.4.2 Dissociation of Enteric Neurospheres into Single Cells
			3.4.3 Expansion of Enteric Neural Stem/Progenitor Cells (See Note 8)
			3.4.4 Neuronal Differentiation of Enteric Neural Stem/Progenitor Cells
	4 Notes
	References
Chapter 7: Preparation of Neural Stem Cells and Progenitors: Neuronal Production and Grafting Applications
	1 Introduction
		1.1 Preface
		1.2 Terminology
		1.3 Neural Stem Cells and Neural Progenitors of the Developing Spinal  Cord
		1.4 Preparation of Fetal Spinal Cord (FSC) Tissue from E13.5-14  Rat
	2 Materials
		2.1 Dissection of FSC Tissue
		2.2 Preparation of FSC Tissue for Transplantation
		2.3 Preparation of Neuronal Restricted Progenitors and Glial Restricted Progenitors from E13.5-14 Rat or from E12.5-13 Mouse S...
			2.3.1 Preparation of Poly-L-Lysine (PLL) and Laminin (LAM)-Coated Dishes for NRP/GRP Culture
			2.3.2 NRP/GRP Medium
			2.3.3 NRP/GRP Dissection
			2.3.4 NRP/GRP Dissociation
			2.3.5 NRP/GRP Culture
			2.3.6 Differentiation of NRPs/GRPs Toward Neuronal Lineage
			2.3.7 Differentiation of NRPs/GRPs Toward Ventral Lineage
			2.3.8 Freezing NRPs/GRPs
			2.3.9 Thawing NRPs/GRPs
		2.4 Characterizing NRP/GRP Cultures In Vitro with Immunocytochemistry
			2.4.1 Staining Live Cells
			2.4.2 Staining Fixed Cells
		2.5 Preparation of Human Neurons from Human Embryonic Stem Cells (hESCs), Induced Pluripotent Stem Cells (hiPSCs) and Direct R...
			2.5.1 Preparation of Dishes and Media for Human Embryonic Stem Cells (hESCs) and Human Induced Pluripotent Cells (hiPSCs) for ...
			2.5.2 Thawing hESCs/hiPSCs
			2.5.3 Monolayer (2D) Differentiation Protocol for hESCs and Human Induced Pluripotent Cells hiPSCs
			2.5.4 Embryoid Body (3D) Differentiation Protocol for hESCs and Human Induced Pluripotent Cells hiPSCs
			2.5.5 Preparation of Dishes and Media for Direct Differentiation of Human Somatic Cells (Fibroblasts/Pericytes) into Cortical ...
			2.5.6 Thawing Fibroblasts/Pericytes
			2.5.7 Direct Reprogramming of Human Skin Fibroblasts/Pericytes into Cortical Glutamatergic Neurons
	3 Methods
		3.1 Dissection of FSC Tissue
		3.2 Preparation of FSC Tissue for Transplantation
		3.3 Preparation of Neuronal Restricted Progenitors and Glial Restricted Progenitors from E13.5-14 Rat or from E12.5-13 Mouse S...
			3.3.1 Preparation of Poly-L-Lysine (PLL) and Laminin (LAM)-Coated Dishes for NRP/GRP Culture
			3.3.2 NRP/GRP Medium
			3.3.3 NRP/GRP Dissection
			3.3.4 NRP/GRP Dissociation
			3.3.5 NRP/GRP Culture
			3.3.6 Differentiation of NRPs/GRPs Toward Neuronal Lineage
			3.3.7 Differentiation of NRPs/GRPs Toward Ventral Lineage
			3.3.8 Freezing NRPs/GRPs
			3.3.9 Thawing NRPs/GRPs
		3.4 Characterizing NRP/GRP Cultures In Vitro with Immunocytochemistry
			3.4.1 Staining Live Cells
			3.4.2 Staining Fixed Cells
		3.5 Preparation of Human Neurons from Human Embryonic Stem Cells (hESCs), Induced Pluripotent Stem Cells (hiPSCs) and Direct R...
			3.5.1 Preparation of Dishes and Media for Human Embryonic Stem Cells (hESCs) and Human Induced Pluripotent Cells (hiPSCs) for ...
			3.5.2 Thawing hESCs/hiPSCs
			3.5.3 Monolayer (2D) Differentiation Protocol for hESCs and Human Induced Pluripotent Cells hiPSCs
			3.5.4 Embryoid Body (3D) Differentiation Protocol for hESCs and Human Induced Pluripotent Cells hiPSCs
			3.5.5 Preparation of Dishes and Media for Direct Differentiation of Human Somatic Cells (Fibroblasts/Pericytes) into Cortical ...
			3.5.6 Thawing Fibroblasts/Pericytes
			3.5.7 Direct Reprogramming of Human Skin Fibroblasts/Pericytes into Cortical Glutamatergic Neurons
		3.6 Summary
	4 Notes
	References
Chapter 8: Slice Culture Modeling of CNS Viral Infection
	1 Introduction
	2 Materials
		2.1 Slice Preparation (See Note 1)
			2.1.1 Brain
			2.1.2 Spinal  Cord
		2.2 Viral Infection
		2.3 Culture Maintenance
		2.4 RT-PCR Quantification of Viral or Host Genes
		2.5 Immunofluorescence Imaging with Cryosectioning
		2.6 Immunofluorescence Imaging Without Cryosectioning
		2.7 Propidium Iodide (PI) Quantification of Tissue Injury
		2.8 Lactate Dehydrogenase (LDH) Quantification of Tissue Injury
		2.9 MTT (3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide) Visualization of Tissue Injury
		2.10 Western Blotting
		2.11 Caspase Activity Assay
		2.12 Cytokine Analysis
		2.13 Pharmaceutical/Cytokine Treatment
	3 Methods
		3.1 Brain and Spinal Cord Tissue Preparation
		3.2 Plating Brain and Spinal Cord Slices
		3.3 Viral Infection
		3.4 Culture Maintenance (See Note 14)
		3.5 RT-PCR Quantification of Viral or Host Genes
			3.5.1 Harvest Slices into RLT-βME Buffer
			3.5.2 Purify Slice  RNA
		3.6 RT-PCR Quantification
		3.7 Immunofluorescence Imaging with Cryosectioning (See Note 16)
			3.7.1 Fix and Cryoprotect Tissue
			3.7.2 Form a ``Face-off´´ TFM Block (Used to Form a Surface that Is Parallel to Cryostat Blade)
			3.7.3 Cryoembed Slices
			3.7.4 Cryosectioning
			3.7.5 Immunohistochemistry
		3.8 Immunofluorescence Imaging Without Cryosectioning
		3.9 Propidium Iodide (PI) Quantification of Tissue Injury
		3.10 Lactate Dehydrogenase (LDH) Quantification of Tissue Injury
		3.11 MTT (3-(4,5- Dimethylthiazol-;2-;yl)-2,5-Diphenyltetrazolium Bromide) Visualization of Tissue Injury
		3.12 Western Blotting
		3.13 Caspase Activity Assay
		3.14 Cytokine Analysis
			3.14.1 Cytokine Screen on Multianalyte ELISArray Plates
			3.14.2 Cytokine Quantification Single-Analyte ELISA Plates
		3.15 Pharmaceutical/Cytokine Treatment
	4 Notes
	References
Chapter 9: Neurospheres and Glial Cell Cultures; from Plating to Cell Phenotyping
	1 Introduction
		1.1 Preface
	2 Materials
		2.1 Tissue Culture Equipment
		2.2 Culture of Neurospheres
		2.3 Culture Media
		2.4 Immunocytochemistry Reagents
	3 Methods
		3.1 Isolation of Neural Stem Cells
		3.2 Passaging of Neurosphere Cultures
		3.3 Differentiation of Neurospheres
			3.3.1 Coat Chamber Slides or Coverslips with Poly-L-Ornithine/Laminin
			3.3.2 Differentiation of Intact Neurospheres
			3.3.3 Differentiation of Dissociated Neurospheres
			3.3.4 Culture Conditions to Preferentially Differentiate Neural Stem Cells into Neurons
			3.3.5 Culture Conditions to Preferentially Differentiate Neural Stem Cells into Astrocytes
			3.3.6 Culture Conditions to Preferentially Differentiate Neural Stem Cells into Oligodendrocytes
	4 Immunocytochemical Analysis of Cells Derived from Differentiated Neurospheres
		4.1 Preparing Paraformaldehyde Fixing Solution
		4.2 Fixation and Permeabilization of Differentiated Cells
			4.2.1 Fixation with Paraformaldehyde
			4.2.2 Fixation with Methanol
			4.2.3 Fixation with Acetone
		4.3 Immunolabeling of Fixed Cells
	5 Immunocytochemical Analysis of Intact, Undifferentiated Neurospheres
		5.1 Fixation and Permeabilization of Intact Neurospheres
	6 Astrospheres Generated from Brain Tumor Stem Cells
		6.1 Coat Tissue Culture Dishes with Poly-HEMA
	7 Notes
	References
Chapter 10: Transfection of Neuronal Cultures
	1 Introduction
	2 Materials
	3 Methods
	4 Notes
	References
Chapter 11: Lentiviral Transduction of Neuronal Cells
	1 Introduction
	2 Materials
	3 Methods
		3.1 Lentiviral Transfection and Production
		3.2 Titration of Lentivirus
			3.2.1 Transduction of Primary Fetal Astrocytes
		3.3 Conclusion
	4 Notes
	References
Chapter 12: Compartmentalized Neuronal Cultures
	1 Materials
		1.1 Brain Tissue
		1.2 Reagents
		1.3 Dissection Instruments, Plasticware, and Glassware (all Sterile)
	2 Methods
		2.1 Preparation of Slides
		2.2 Assembling the AXIS Device on Slides and Preparation for Plating of Cells
		2.3 Plating Neurons
	3 Notes
	References
Chapter 13: Quantitative Assessment of Neurite Outgrowth Over Growth Promoting or Inhibitory Substrates
	1 Introduction
	2 Materials
		2.1 Reagents
		2.2 Neurobasal A Based Culture Medium
		2.3 Animals
		2.4 Preparation of Coverslips
		2.5 Preparation of Permissive Substrate
		2.6 Preparation of Inhibitory Substrate
		2.7 Percoll Gradient Preparation
	3 Methods
		3.1 Dissection and Dissociation
		3.2 Separation of Cells by Density Gradient Centrifugation
		3.3 Immunocytochemical Staining
		3.4 Image Capture
		3.5 Measurements of Neurite Length
	4 Notes
	References
Chapter 14: Isolation and Culture of Dorsal Root Ganglia (DRG) from Rodents
	1 Introduction
	2 Materials and Reagents
		2.1 Growth Media
	3 Methods
		3.1 Coating Coverslips and Plates
		3.2 Dissection of DRGs from Adult Mouse
		3.3 Dissection of DRGs from Embryonic Mouse or Rat
		3.4 DRG Tissue Digestion and Culture
	4 Notes
	References
Chapter 15: Isolation of Primary Human and Rodent Brain Microvascular Endothelial Cells: Culturing, Characterization, and High...
	1 Introduction
	2 Materials
	3 Methods
		3.1 Cell Culture
		3.2 Transfection
		3.3 Microscopy
		3.4 Antibodies
		3.5 Immunocytochemistry
		3.6 Conclusions
	4 Notes
	References
Chapter 16: Fetal Brain Injury Models of Fetal Alcohol Syndrome: Examination of Neuronal Morphologic Condition Using Sholl Ass...
	1 Introduction
	2 Materials
	3 Methods
		3.1 Cell Culture
		3.2 Treatment
		3.3 Transfection
		3.4 Sholl Analysis
		3.5 Microscopy
		3.6 Conclusion
	4 Notes
	References
Index