Methods in Molecular Biology
Chapter 1: Neuronal Cell Death: An Overview of Its Different Forms in Central and Peripheral Neurons
1 Brief History of Neuronal Cell Death
1.1 Discovery of Cell Death
1.2 Cell Death in the Nervous System
2 Morphological Types of Cell Death in Neurons
3 Neuronal Cell Death Pathways and Their Recognition in Death Assays
4 Concluding Remarks and Future Perspectives
Part I: Molecular, Structural, Functional, and Genomic Changes in Dying Neurons
Chapter 2: Cell Volume Regulation Monitored with Combined Epifluorescence and Digital Holographic Microscopy
1.1 Principle of Quantitative Phase Measurement
1.2 Phase Signal Interpretation
1.3 Ratiometric Fluorescence
2.2 Cell Culture Handling
2.4 Loading of Calcium Indicator
Chapter 3: Flow Cytometric Analysis of DNA Synthesis and Apoptosis in Central Nervous System Using Fresh Cell Nuclei
2.2 Equipment and Software
3.1 Active Caspase 3 Immunolabeling and Detection
3.3 EdU Incorporation Assay and Detection
Chapter 4: Nuclear Signs of Pre-neurodegeneration
2.2 Cellular Dissociation Squash Technique
2.3 Immunoưfluorescence Cell Staining
2.4 Imaging Nuclear Signs of Pre-neurodegeneration
3.2 Cellular Dissociation Squash Technique
3.3 Immunoưfluorescence Cell Staining
3.4 Imaging Nuclear Signs of Pre-ưneurodegeneration
Chapter 5: Multi-parametric O2 Imaging in Three-Dimensional Neural Cell Models with the Phosphorescent Probes
2.4.1 Preparation of PDL-Coated Slices
3.1 Neurosphere Culture and Staining with Imaging Probes
3.2 Brain Slice Culture and Staining with Imaging Probes
3.3 Imaging and Data Processing
Chapter 6: Calcium Imaging in Neuron Cell Death
2.1 Primary Culture of Rat Hippocampal Neurons
2.3 Excitotoxicity and Apoptosis Assays
3.1 Primary Rat Hippocampal Neuron Cell Culture
3.2 Fluorescence Imaging of Cytosolic Ca2+
3.3 Excitotoxicity and Apoptosis Assessment
3.4 Combination of Cytosolic Ca2+ Imaging and Annexin V Staining
Chapter 7: Monitoring Mitochondrial Membranes Permeability in Live Neurons and Mitochondrial Swelling Through Electron Microscopy Analysis
2.1 Mitochondrial Permeability Transition by Live Cell Imaging
2.2 Mitochondrial Swelling Detection from Electron Microscopy Analysis
3.1 Mitochondrial Permeability Transition by Live Cell Imaging
3.2 Mitochondrial Swelling Detection from Electron Microscopy Analysis
Chapter 8: Real-Time Visualization of Caspase-3 Activation by Fluorescence Resonance Energy Transfer (FRET)
2.1 Preparation of Cerebellar Organotypic Cultures (OCCs)
2.3 Imaging of OCCs and FRET Measurements
3.2.1 Heat-Shock Transformation to Produce Plasmid-DNA
3.2.2 Plasmid DNA Extraction and Concentration
3.2.3 Gene Gun Cartridges Preparation
3.3 Imaging of OCCs and FRET Measurements
3.3.1 LSCFM on Fixed Cells
3.3.2 LSCFM on Live Cells
3.3.3 Preliminary Evaluation of FRET Efficiency
3.3.4 Measuring Caspase-3 Activity in Fixed OCCs
3.3.5 Measuring Caspase-3 Activity in Live OCCs
Chapter 9: Design and Cloning of Short Hairpin RNAs (shRNAs) into a Lentiviral Silencing Vector to Study the Function of Selected Proteins in Neuronal Apoptosis
2.1 Design, Production, and Cloning of shRNAs and Preparation of Lentiviral Vectors
2.2 Primary Cerebellar Granule Neuron Culture, Induction, and Detection of Apoptosis
2.3 Western Blotting and Immuno-fluorescence for d-Serine Racemase
3.1 Design, Production, and Cloning of shRNAs and Preparation of Lentiviral Vectors
3.1.2 Annealing of shRNA Oligonucleotides
3.1.3 Cloning ShRNA Oligonucleotides into pLVTHM
3.1.4 Preparation of Lentiviral Vectors
3.2 Primary Cerebellar Granule Neuron Cultures and Lentivirus Transduction
3.2.1 Primary Cerebellar Granule Neuron Cultures
3.2.2 Transduction of Primary Cerebellar Granule Neuron Cultures
3.3 Induction and Detection of Apoptosis
3.3.1 Induction of Apoptosis
3.3.2 Detection of Apoptosis
3.4 Western Blotting and Immuno-fluorescence for d-Serine Racemase
3.4.2 Immuno-fluorescence
Chapter 10: Genomic Analysis Using Affymetrix Standard Microarray GeneChips (169 Format) in Degenerate Murine Retina
2.2.1 RNA Extraction and DNAse Treatment
2.2.2 cDNA Synthesis and Amplification
2.2.3 Fragmentation and Labeling
2.2.4 Hybridization, Washing, Staining, and Scanning
3.1 Thermo Cycler Programs
3.2 Hybridization Oven Program
3.4 cDNA Synthesis and Amplification
3.5 Fragmentation and Labeling
Chapter 11: Genomic Analysis of Transcriptional Changes Underlying Neuronal Apoptosis
2.1 General and Safety Notes
2.3 Equipment and Reagents
3.2.2 cRNA Synthesis and Amplification
Chapter 12: High-Throughput Cell Death Assays
2.1 Cell Culture Media and Treatment Media Components
2.2 Other Materials and Hardware Components
3.1 Preparation of Culture Media and Cell Cultures
3.2 Seeding Cells onto Microplates
3.3 Treating Cells for Experimentation
3.4 Data Acquisition and Analysis
Part II: Cell Death in Neuropathology and Experimental Neuropathology
Chapter 13: Staining of Dead Neurons by the Golgi Method in Autopsy Material
3.1 Semi-rapid Golgi Method
3.2 Morphology and Quantitation
3.2.2 Diencephalon, Encephalic Trunk, and Cerebellum
Chapter 14: Image Analysis Algorithms for Immunohistochemical Assessment of Cell Death
2.1 Specimens, Buffers, Histology or IHC Reagents and Detection Systems
2.1.2 Anesthesia and Perfusion Buffers
2.2 Immunohistoưchemical Reagents and Detection Kits
2.3 Special Instrumentation and Equipment
3.1 Artifact-Free Preparation of Brains for IMF on Fixed-Frozen Cryosections (FFC) or Immunoưhistochemical Applications Using Tissue Sections from Z-Fix-Fixed Paraffin-Embedded (ZFPE) Blocks (See Note 5)
3.2 IMF on FFCs or HRP-IHC on ZFPE Sections
3.3 Quantitative Analysis
3.4 Assessment of Brain Lesion Volume
3.5 Neuropathoưlogical Scoring of Brain Injury
Chapter 15: In Vitro Oxygen-Glucose Deprivation to Study Ischemic Cell Death
2.1 Media for OGD in Primary Neurons and Organotypic Slices
2.2 Buffers for OGD in Acute Slices
2.3.3 Primary Neurons and Organotypic Slices
2.4 Cell Death and Viability Assays
3.1 OGD in Primary Neurons and Organotypic Slice Cultures
3.2 OGD in Acute Hippocampal Slices
3.3 Assessment of Cellular Damage
3.3.1 PI Uptake in Primary Neurons
3.3.2 PI Uptake in Slices
Chapter 16: Laser Microbeam Targeting of Single Nerve Axons in Cell Culture
2.2 RoboLase: Optical Design and Hardware
2.2.1 Individual Components
2.2.2 External Laser Optics and Hardware
3.1 Primary Nerve Cell Preparation
3.2 Laser Alignment and Targeting
3.2.2 Laser Power Measurement
3.2.3 Laser Dosage Calculation
3.4 Nerve Regeneration: Live Cell Imaging
Chapter 17: Real-Time Imaging of Retinal Cell Apoptosis by Confocal Scanning Laser Ophthalmoscopy
2.2 Drugs and Other Materials
3.2 Administration of Fluorescent Annexin A5
3.2.1 Intravitreal Injection (See Note 4)
3.2.2 Intravenous Injection (See Note 4)
3.3 Adaptation of the cSLO for Small Animal Imaging
3.5 Image Acquisition (Fig. 5)
Chapter 18: Targeted Toxicants to Dopaminergic Neuronal Cell Death
2.1 Treatment of Primary Mouse Mesencephalic Cultures with MPP+
2.2 Tyrosine Hydroxylase (TH) Immunocytochemistry of Primary Cultures
2.3 Treatment of Intact Animals with MPTP
2.4 TH Immunohisto-chemistry of Intact Animals
3.1 Treatment of Primary Mouse Mesencephalic Cultures with MPP+
3.2 TH Immunocytochemistry of Primary Mesencephalic Cultures
3.3 Treatment of Intact Animals with MPTP
3.4 TH Immunohisto- chemistry of Intact Animals
Part III: Neural Stem Cells, Progenitors, and Gene Therapy Strategies
Chapter 19: Stem Cells, Neural Progenitors, and Engineered Stem Cells
2.1 Human Pluripotent Stem Cell Passaging and Subculture
2.2 Derivation of Human Neural Progenitors from Human Pluripotent Stem Cells
2.3 Passage and Subculture of Human Neural Progenitors
2.4 Dideoxycytidine (ddC) Treatment of Human Neural Progenitors
2.5 Isolation and Introduction of Pathogenic mtDNA into ddC-Human Neural Progenitors
2.6 Labeling Pathogenic mtDNA and rhTFAM
2.8 Restriction Enzyme Digestion
2.9 Differentiation of Engineered Human Neural Progenitors into Neurons
3.1 Human Pluripotent Stem Cell Passaging and Subculture
3.2 Derivation of Human Neural Progenitors from Human Pluripotent Stem Cells
3.3 Passage and Subculture of Human Neural Progenitors
3.4 Dideoxycytidine (ddC) Treatment of Human Neural Progenitors
3.5.2 Localization of Specific Markers in Human Neural Progenitors by Immunocytochemical Analysis
3.6 Preparation of LHON Pathogenic mtDNA and Introduction of LHON Pathogenic mtDNA into ddC-Treated Human Neural Progenitors
3.7 Imaging of LHON mtDNA Entry into ddC-Treated Human Neural Progenitors
3.8 Restriction Enzyme Digestion Analysis
3.9 Differentiation Potential of Engineered hNP Cells into Neurons
Chapter 20: Herpes Simplex Virus Type 1 (HSV-1)-Derived Recombinant Vectors for Gene Transfer and Gene Therapy
1.1 Herpes Simplex Virus Type 1 and Its Derived Vectors
1.1.2 HSV-1-Derived Vectors
1.1.3 Replication-ưDefective Recombinant HSV-1 Vectors
1.1.4 Attenuated Recombinant HSV-1 Vectors
1.2 Construction of Recombinant HSV-1 Vectors by Homologous Recombination in Eukaryotic Cells
1.3 Construction of Recombinant HSV-1 Vectors by Homologous Recombination in Bacteria: ET Recombination and GalK-ưPositive/Negative Selection
2.1 Construction of Recombinant HSV-1 Vectors by Homologous Recombination in Eukaryotic Cells
2.1.1 Materials and Solutions for Viral Stock Preparation and OptiPrep Gradient
2.2 Construction of Recombinant HSV-1 Vectors by Homologous Recombination in Bacteria
2.2.1 Generation of a Targeting DNA Fragment by PCR Amplification
2.2.2 Preparation of Electrocompetent E. coli, Electroporation, and Selection of GalK-Positive and GalK- Negative Bacteria
2.2.3 Isolation and Analysis of BAC DNA from E. coli (Miniprep Protocol)
2.2.4 Transfection of Mammalian Cells with BAC DNA and Reconstitution of Recombinant HSV-1
3.1 Construction of Recombinant HSV-1 Vectors by Homologous Recombination in Eukaryotic Cells
3.1.1 Preparation of Viral DNA for Transfection
3.1.2 Co-transfection of Plasmid DNA and Viral DNA to Generate a Recombinant Virus
3.1.3 Recombinant Virus Isolation by Limiting Dilution
3.1.4 Preparation of High-Titer Replication-ưDefective Recombinant Viral Stock
3.1.5 Titration of Virus Stock
3.1.6 Purification of Recombinant HSV-1 Stock
3.1.7 Collection of Virus Particles
3.2 Construction of Recombinant HSV-1 Vectors by Homologous Recombination in Bacteria
3.2.1 Generation of a GalK+ Targeting DNA Fragment by PCR Amplification
3.2.2 Preparation of Electrocompetent E. coli SW102
3.2.3 Electroporation of HSV-1 BAC DNA into E. coli SW102
3.2.4 Electroporation of the GalK Targeting DNA into E. coli SW102 Containing the HSV-1 BAC and Screening for GalK-ưPositive Clones (galK-ưPositive Selection)
3.2.5 Electroporation of the Targeting DNA into Galk-Positive E. coli SW102 Containing the HSV-1 BAC and Screening for Galk-ưNegative Clones (Galk-ưNegative Selection)
3.2.6 Isolation and Characterization of HSV-BAC DNA from Small Bacterial Cultures
3.2.7 Transfection of Mammalian Cells with BAC DNA and Reconstitution of Recombinant HSV-1
Chapter 21: Herpes Simplex Virus Type 1 (HSV-1)-Derived Amplicon Vectors for Gene Transfer and Gene Therapy
1.1 HSV-1-Based Amplicon Vectors
1.2 Preparation of Non-toxic Amplicon Vector Stocks
1.2.1 Production of Amplicon Vectors by Co-transfecting Amplicon Plasmids DNA and Helper Genomes
1.2.2 Production of Amplicon Vectors Using the Cre/Loxp1 Site-Specific Recombination System
2.1 Packaging of HSV-1 Amplicon Vectors Using a Replication-ưCompetent, Packaging-ưDefective HSV-1 Genome Cloned as a BAC
2.1.1 Preparation of HSV-1 BAC DNA
2.1.2 Preparation of HSV-1 Amplicon Vector Stocks
2.1.3 Harvesting, Purification, and Titration of HSV-1 Amplicon Vectors
2.2 Packaging of Amplicon Vectors Using a Replication-ưIncompetent, Cre/loxP1 Sensitive Helper Virus
2.2.1 Preparation of the Defective Helper Virus
3.1 Packaging of HSV-1 Amplicon Vectors Using a Replication-ưCompetent, Packaging-ưDefective HSV-1 Genome Cloned as a BAC
3.1.1 Preparation of HSV-1 BAC DNA
3.1.2 Preparation of Plasmid DNA (Maxiprep Protocol–Qiagenđ)
3.1.3 Transfection of Vero 2-2 Cells and Harvesting, Concentration, and Purification of Packaged Amplicon Vectors
3.1.4 Titration of HSV-1 Amplicon Vector Stocks
3.2 Packaging of Amplicon Vectors Using a Replication-ưIncompetent, Cre/loxP1-ưSensitive Helper Virus
3.2.1 Production, Purification, and Titration of HSV-1-LaLΔJ Helper Virus Stocks
3.2.2 Production of Amplicon Vectors Using Cre/loxP1 Site-Specific Recombination
Generation of P0 Stock (Helper-Contaminated, HC)
Titration of Amplicon Vectors and Helper Virus in P0 Stocks
Amplification from P0 to P1 and Titration of P1 Stocks (Helper-Contaminated)
Amplification from P1 to P2 and Titration of P2 Stocks (Helper-Contaminated)
Production and Titration of P3 Amplicon Vector Stocks (Helper-Free, HF)
Chapter 22: Bone Marrow Transplantation for Research and Regenerative Therapies in the Central Nervous System
2.2 Bone Marrow Stem Cell Extraction
3.2 Bone Marrow Stem Cell Extraction
Part IV: Neuronal Death in Nonmammalian Models
Chapter 26: Drosophila Model for Studying Phagocytosis Following Neuronal Cell Death
3.2.2 Imaging of Fixed Embryos
3.3.1 Preparatory Steps for Injection
3.3.3 Time Lapse Recording
Chapter 23: Detection of Activated Caspase-8 in Injured Spinal Axons by Using Fluorochrome-Labeled Inhibitors of Caspases (FLICA)
2.3 Solutions and Fixatives
3.2 Spinal Cord Transection
3.3 Detection of Activated Caspase-8 in Axons of Whole-ưMounted Ex-Vivo Tissue
Chapter 24: Generation of Zebrafish Models by CRISPR/Cas9 Genome Editing
3.1 Oligo Design for gRNA Transcription
3.2 RNAsecure Treatment (See Note 4) and Oligo Annealing
3.3 gRNA In Vitro Transcription (MEGAshortscript T7 Transcription Kit)
3.4 Cas9 Plasmid Preparation
3.5 Cas9 In Vitro Transcription (mMessage mMachine SP6 Transcription Kit)
3.8 PCR and Restriction Fragment Length Polymorphisms (RFLP) Analysis
3.9 Screen for Inherited Genome Modifications
Chapter 25: In Vivo Assessment of Neuronal Cell Death in Drosophila
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