Programmed Cell Death: Methods and Protocols

✍ Scribed by Hamsa Puthalakath, Christine J. Hawkins

• Publisher: Humana Press
• Year: 2016
• Tongue: English
• Leaves: 294
• Series: Methods in Molecular Biology; 1419
• Category: Library

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: Detection of Apoptotic Versus Autophagic Cell Death by Flow Cytometry
1 Introduction
2 Materials
2.1 Disposables and Equipment
2.2 Cell Maintenance
2.3 RNA Interference
2.4 Pharmacological Treatments and DiOC6(3)/PI Co-staining
3 Methods
3.1 Cell Maintenance
3.2 RNA Interference
3.3 Pharmacological Treatments and DiOC6(3)/PI Co-staining
4 Notes
References
Chapter 2: In Vivo Apoptosis Imaging Using Site-Specifically 68Ga-Labeled Annexin V
1 Introduction
2 Materials
2.1 Equipment
2.2 Solutions
2.3 Animals
3 Methods
3.1 Radiosynthesis
3.2 PET Imaging
4 Notes
References
Chapter 3: Detection of Active Caspases During Apoptosis Using Fluorescent Activity-Based Probes
1 Introduction
2 Materials
2.1 Components for Labeling Caspases with Activity-­Based Probes
2.2 Components for Fluorescent SDS-PAGE
2.3 Immuno-precipitation Components
2.4 Components for In Vivo Detection of Caspases
3 Methods
3.1 Labeling of Active Caspases in Cell Lysates
3.2 Labeling of Active Caspases in Intact/Dying Cells
3.3 Detection of Probe-Labeled Caspases by Fluorescent SDS-PAGE
3.4 Immuno-precipitations of Probe-Labeled Lysates to Confirm ABP Targets
3.5 In Vivo Detection of Caspase Activity
4 Notes
References
Chapter 4: Detection of Initiator Caspase Induced Proximity in Single Cells by Caspase Bimolecular Fluorescence Complementation
1 Introduction
2 Materials
2.1 Plasmids
2.2 Cell Culture and Transfection
2.3 Induction of Apoptosis
2.4 Microscope Instrumentation
2.5 Data Processing
3 Methods
3.1 Transfection of Caspase BiFC Plasmids
3.2 Induction of Apoptosis
3.3 Caspase BiFC for Single Time Point Data Acquisition
3.4 Caspase BiFC for Time-Lapse Microscopy
3.5 Data Analysis
3.5.1 Calculate the Percentage of Venus-Positive Cells at a Single Time Point
3.5.2 Calculate the Intensity of Venus-Positive Cells Over Time
4 Notes
References
Chapter 5: In Vitro Use of Peptide Based Substrates and Inhibitors of Apoptotic Caspases
1 Introduction
2 Materials
3 Methods
3.1 Preparation of Cytosolic Extract for Apoptosome Activation
3.2 Activation of Apoptosome in Cytosolic Extracts
3.3 Measuring Caspase Activity Using Fluorogenic Peptide Substrates
3.4 Immunopre cipitation of Caspases from Activated Cytosolic Extracts to Demonstrate Activity of Each Caspase with Each Substrate
3.5 Use of Peptide-­Based Inhibitors in Apoptosome Activation Assays: Inhibition Prior to Apoptosome Activation
3.6 Use of Peptide-­Based Inhibitors in Apoptosome Activation Assays: Inhibition After Apoptosome Activation
4 Notes
References
Chapter 6: Experimental In Vivo Sepsis Models to Monitor Immune Cell Apoptosis and Survival in Laboratory Mice
1 Introduction
2 Materials
2.1 Induction of Polymicrobial Sepsis by Cecal Ligation and Puncture (CLP) or Cecal Slurry Injection
2.2 “Second Hit” Intranasal Infection and Intra-­Tracheal Intubation
3 Methods
3.1 Cecal Ligation and Puncture (CLP)
3.2 Cecal Slurry (CS) Injection
3.2.1 Preparation of Cecal Slurry
3.2.2 Injection of Cecal Slurry
3.3 Assessment of Immune Cell Apoptosis in Septic Mice
3.4 “Second Hit” Infection with Pseudomonas aeruginosa
3.4.1 Microbiologic Preparation of Pseudomonas aeruginosa
3.4.2 Intratracheal Intubation and Infection
3.4.3 Intra-Nasal Infection
3.4.4 Sepsis Monitoring Sheet
4 Notes
References
Chapter 7: Analysis of Cell Death Induction in Intestinal Organoids In Vitro
1 Introduction
2 Materials
3 Methods
3.1 Crypt Isolation
3.2 Apoptosis Induction and Detection
4 Notes
References
Chapter 8: In Vitro Differentiation of Mouse Granulocytes
1 Introduction
2 Materials
2.1 Production of Inducible Hoxb8 Lentiviral Particles
2.2 Isolation of Lineage Marker Negative Cells
2.3 Generation and Cultivation of Inducible Hoxb8 Immortalized Cells
3 Methods
3.1 Production of Inducible Hoxb8 Lentiviral Particles
3.2 Isolation of Lineage Marker Negative Cells (See Note 2)
3.3 Generation and Cultivation of Inducible Hoxb8-­Immortalized Cells
3.4 In Vitro Differentiation of SCF-condHoxb8 Cells into Mature Neutrophils
3.5 In Vitro Differentiation of IL-3-condHoxb8 Cells into Mature Basophils
4 Notes
References
Chapter 9: Hydrodynamic Injection as a Method of Gene Delivery in Mice: A Model of Chronic Hepatitis B Virus Infection
1 Introduction
2 Materials
2.1 Preparation of HBV Plasmid
2.2 Hydrodynamic Injection
3 Methods
3.1 Preparation of HBV Plasmid
3.2 Hydrodynamic Injection
4 Notes
References
Chapter 10: Isolation of Cardiomyocytes and Cardiofibroblasts for Ex Vivo Analysis
1 Introduction
2 Materials
2.1 Material for Isolation of Neonatal Mouse Cardiomyocytes
2.2 Materials for Immortalization of Neonatal Mouse Cardiofibroblasts
2.3 Material for Isolation and Culture of Primary Adult Mouse Cardiomyocytes
3 Methods
3.1 Method for Isolation of Neonatal Mouse Cardiomyocytes and Cardiofibroblasts
3.2 Immortalization of Neonatal Mouse Cardiofibroblasts With SV40 T-Antigen
3.3 Isolation and Culture of Primary Adult Mouse Cardiomyocytes
3.3.1 Harvesting of the Mouse Heart
3.3.2 Heart Cannulation, Perfusion and Enzymatic Digestion
4 Notes
References
Chapter 11: Detection of Cell Death in Drosophila Tissues
1 Introduction
2 Materials
3 Methods
3.1 Tools for Manipulating Cell Death
3.1.1 Genetic Tools for Blocking or Inducing Cell Death in Drosophila
3.1.2 Genotoxic Methods to Induce Cell Death
3.2 Detecting Cell Death
3.2.1 Collecting Samples of Defined Ages for Analysis
3.2.2 TUNEL Assay
3.2.3 Immunostaining of Apoptotic Cells
3.2.4 Acridine Orange Staining
3.3 Bioch mical Assays
3.3.1 Colorimetric Assay for Caspase Activity
3.3.2 Viability Assays in S2 Cells
4 Notes
References
Chapter 12: Methods to Study Plant Programmed Cell Death
1 Introduction
2 Materials
2.1 Growth of Lace Plant and Live Cell Imaging of Developmental PCD
2.2 Growth of Ryegrass Seedlings and the Root Hair Assay for Quantification of Rates of PCD
2.3 Growth of Arabidopsis Plants, PCD Induction, and Leaf Discs ion Leakage Measurements
3 Methods
3.1 Studying Developmental PCD in Lace Plant Leaves
3.2 Quantitative Determination of Abiotic Stress Induced PCD in Ryegrass Root Hairs
3.3 Determination of Cellular Damage Using ion Leakage
4 Notes
References
Chapter 13: Modeling Metazoan Apoptotic Pathways in Yeast
1 Introduction
1.1 Researching Cell Death Pathways Using Yeast
1.2 Pathways Controlling Caspase-­Dependent Yeast Death
1.3 Pathways Controlling Bax- or Bak-­Dependent Yeast Death
1.4 Transcriptional Reporter System for Testing Caspase Specificity
2 Materials
2.1 Yeast Strains
2.2 Plasmids
2.3 Media
2.4 Other Reagents
2.5 Equipment
3 Methods
3.1 Lithium Acetate Transformation
3.2 Extracting Library Plasmid DNA from Yeast
3.3 Visualizing Caspase/Bax/Bak Activity and Inhibition in Yeast, by Semi-­Quantitative Spotting onto Agar Plates
3.4 Visualizing Activity of BH3-­Mimetics or Small Molecule Caspase Inhibitors in Yeast
3.5 Defining the Minimal Specificity of a Protease Using Yeast
3.6 Quantitative Assessment of β-Galactosidase Activity, as a Readout of Caspase Cleavage Efficiency
4 Notes
References
Chapter 14: Characterizing Bcl-2 Family Protein Conformation and Oligomerization Using Cross-Linking and Antibody Gel-Shift in Conjunction with Native PAGE
1 Introduction
2 Materials
2.1 Cell Fractionation Reagents
2.2 Disulfide and Cross-­linking Reagents
2.3 Antibody Reagents for Gel-Shift
2.4 Blue Native PAGE Reagents
2.5 Clear Native PAGE Reagents
2.6 Electrotransfer Reagents
3 Methods
3.1 Cell Fractionation
3.2 Induction of Cysteine Linkage
3.3 Antibody Gel-Shift
3.4 Preparation of Samples for Native PAGE
3.5 Blue Native PAGE [20] (See Fig. 1a)
3.6 High Resolution Clear Native PAGE (See Note 13) [21] (See Fig. 1b)
3.7 Electrotransfer and Immunoblotting
4 Notes
References
Chapter 15: Using Förster-Resonance Energy Transfer to Measure Protein Interactions Between Bcl-2 Family Proteins on Mitochondrial Membranes
1 Introduction
2 Materials
2.1 Purification of Bcl-2 Family Recombinant Proteins
2.2 Labeling Proteins with Donor and Acceptor Maleimide ALEXA Fluorophores
2.3 Mitochondria Preparation
2.4 Cytochrome c Release
2.5 cBID–BCL-XL FRET Interaction in the Presence of Purified Mitochondria
3 Methods
3.1 Purification of Bcl-2 Family Recombinant Proteins
3.2 Labeling of Proteins with Donor and Acceptor Maleimide ALEXA Fluorophores
3.3 Mitochondria Preparation
3.4 Cytochrome c Release
3.5 Measuring the Interaction Between cBID and BCL-XL in Mitochondria Using FRET
4 Notes
References
Chapter 16: Preparing Samples for Crystallization of Bcl-2 Family Complexes
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 17: Screening Strategies for TALEN-Mediated Gene Disruption
1 Introduction
2 Materials
2.1 Maintenance of Cells in Culture, Transfection of TALENs, and Sorting of Transfected Cells
2.2 General Molecular Biology Reagents (Required for All Strategies)
2.3 Heteroduplex Mismatch Assay
2.4 SDS-PAGE and Western Blotting
3 Methods
3.1 Primer Design and Basic PCR Amplification of the Target Site
3.2 Heteroduplex Mismatch Assay
3.3 Transfection of TALENs and Sorting of Transfected Cells
3.4 Expansion of Candidate Gene Disruption Clones
3.5 Freezing Candidate Clones in a 96-Well Plate
3.6 Screening by SDS-PAGE and Western Blot
3.7 Screening by Restriction Digestion
3.8 Sequencing the PCR Product as a Screening Strategy
3.9 Sequencing of Individual Alleles
4 Notes
References
Chapter 18: Using CRISPR/Cas9 Technology for Manipulating Cell Death Regulators
1 Introduction
2 Materials
2.1 Vectors and sgRNA Cloning
2.2 Virus Production and Transduction
2.3 Validation of Knock Out Cell Lines
2.4 Functional Assays
3 Methods
3.1 Designing sgRNAs
3.2 Cloning of sgRNAs into the dox-­Inducible pFgh1tUTG Lentiviral Vector
3.3 Virus Production and Transduction of Cells
3.4 Flow Cytometric Analysis, dox Induction and Sorting of Cells
3.5 Validation of Knock Out Cell Lines
4 Notes
References
Chapter 19: Lentiviral Vectors to Analyze Cell Death Regulators
1 Introduction
2 Materials
2.1 Plasmids
2.2 Cell Culture and Transfection
2.3 Lentivirus Production and Harvest
2.4 Transduction of Target Cells
2.5 Selection of Infected Cells
2.6 Selection of Single Cell Clones
2.7 Testing of Single Cell Clones (Inducible Gene Expression)
3 Methods
3.1 Lentiviral Production
3.2 Transduction of the Target Cell Line
3.2.1 Using Adherent Cells, e.g., MEFs
3.2.2 Using Suspended Cells, e.g., THP-1 Cells
3.3 Selection of Infected Cells
3.3.1 Using Adherent Cells, e.g., MEFs
3.3.2 Using Suspended Cells, e.g., THP-1 Cells
3.4 Isolation of Single Cell Clones
3.4.1 Using Adherent Cells, e.g., MEFs
3.4.2 Using Suspended Cells, e.g., THP-1 Cells
3.5 Testing of Single Cell Clones (Inducible Gene Expression)
4 Notes
References
Chapter 20: Proteomic Profiling of Cell Death: Stable Isotope Labeling and Mass Spectrometry Analysis
1 Introduction
2 Materials
2.1 Reagents
2.2 Equipment
2.3 Buffers and Reagent Preparation
3 Methods
3.1 Preparation of SILAC Media (Triplex Labeling Optional)
3.2 Adaptation of Cells from Normal to SILAC Media
3.3 Sample Preparation of Cell Lysates
3.4 Mass Spectrometry, Peptide Identification, and Protein Quantitation
4 Notes
References
Index