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Cell death techniques : a laboratory manual / edited by Ricky W. Johnstone, John Silke. -- Cold Spring Harbor, New York : Cold Spring Harbor Laboratory Press, c2015. – (58.1574/C393n) |
Contents
Acknowledgments
CHAPTER 1 In
the Midst of Life--Cell Death: What Is It, What Is It Good for, and How to
Study It
INTRODUCTION
In the Midst of Life--Cell Death: What Is It, What
Is It Good for, and How to Study It
CHAPTER 2 Presentation
and Interpretation of Figures in Cell Death Research Publications
INTRODUCTION
Presentation and Interpretation of Figures in Cell
Death Research Publications
PROTOCOLS
1 Preparing
Figures from Cell Death Experimental Data for Publication
2
Interpreting Cell Death Data from Figures in Published Papers
CHAPTER 3 Dead
Cert: Measuring Cell Death
INTRODUCTION
Dead Cert:
Measuring Cell Death
PROTOCOLS
1 Triggering
Apoptosis in Hematopoietic Cells with Cytotoxic Drugs
2 Triggering
Death of Adherent Cells with Ultraviolet Radiation
3 Measuring
Cell Death by Trypan Blue Uptake and Light Microscopy
4 Measuring
Cell Death by Propidium Iodide Uptake and Flow Cytometry 42
5 Measuring
Survival of Adherent Cells with the Colony-Forming Assay
6 Measuring
Survival of Hematopoietic Cancer Cells with the Colony-Forming Assay in Soft Agar
7
Morphological Analysis of Cell Death by Cytospinning Followed by Rapid
Staining
8 Analyzing
Cell Death by Nuclear Staining with Hoechst 33342
9 Detection
of DNA Fragmentation in Apoptotic Cells by TUNEL
10 Measuring
the DNA Content of Cells in Apoptosis and at Different Cell-Cycle Stages by
Propidium Iodide Staining and Flow Cytometry
11 Quantitation of Apoptosis and Necrosis by
Annexin V Binding, Propidium Iodide Uptake, and Flow Cytometry
12 Detecting
Cleaved Caspase
13 Analysis
of Cytochrome c Release by Immunocytochemistry
14 Measuring
Mitochondrial Transmembrane Potential by TMRE Staining
CHAPTER 4 Biochemical
Analysis of Initiator Caspase-Activating Complexes: The Apoptosome and
Death-Inducing Signaling Complex
INTRODUCTION
Biochemical
Analysis of Initiator Caspase-Activating Complexes: The Apoptosome and
Death-Inducing Signaling Complex
PROTOCOLS
2
Activation, Isolation, and Analysis of the Death-Inducing Signaling
Complex 107
CHAPTER 5 Biochemical
Assays of the Proapoptotic Proteins Bak and Bax
INTRODUCTION
Biochemical Assays of the Proapoptotic Proteins Bak
and Bax
PROTOCOLS
1
Investigating Bax Subcellular Localization and Membrane Integration
2
Investigating Bak/Bax Activating Conformation Change by
Immunoprecipitation
3 Detection
of Bak/Bax Activating Conformation Change by Intracellular Flow Cytometry
4
Investigating the Oligomerization of Bak and Bax during Apoptosis by Cysteine
Linkage
5 Blue Native PAGE and Antibody Gel Shift to
Assess Bak and Bax Conformation Change and Oligomerization
CHAPTER 6 Imaging-Based
Methods for Assessing Caspase Activity in Single Cells
INTRODUCTION
Imaging-Based Methods for Assessing Caspase
Activity in Single Cells
PROTOCOLS
1 Measuring
Initiator Caspase Activation by Bimolecular Fluorescence Complementation
2 Measuring
Caspase Activity by F6rster Resonance Energy Transfer
CHAPTER 7 Measuring
Apoptosis: Caspase Inhibitors and Activity Assays
INTRODUCTION
Measuring Apoptosis: Caspase Inhibitors and
Activity Assays
PROTOCOLS
1
Preparation of Cytosolic Extracts and Activation of Caspases by
Cytochrome c
2 Assaying
Caspase Activity In Vitro
3 Detection
of Caspase Activity by Using Antibody-Based Techniques
4 Identification
of Active Caspases by Using Affinity-Based Probes
5 Verification
of a Putative Caspase Substrate
CHAPTER 8 Methods
for Probing Lysosomal Membrane Permeabilization
INTRODUCTION
Methods for Probing Lysosomal Membrane Permeabilization
PROTOCOLS
1 Quantification of Lysosomal Membrane
Permeabilization by Cytosolic Cathepsin and β-N-Acety1-Glucosaminidase
Activity Measurements
2
Visualizing Lysosomal Membrane Permeabilization by Fluorescent Dextran
Release
3 A Method
to Monitor Lysosomal Membrane Permeabilization by Immunocytochemistry
CHAPTER 9 Strategies
for Assaying Lysosomal Membrane Permeabilization
INTRODUCTION
Strategies for Assaying Lysosomal Membrane
Permeabilization
PROTOCOLS
1 The Use of
Lysosomotropic Dyes to Exclude Lysosomal Membrane Permeabilization
2 Measuring
Cysteine Cathepsin Activity to Detect Lysosomal Membrane Permeabilization
3 Studying
Lysosomal Membrane Permeabilization by Analyzing the Release of Preloaded
BSA-Gold Particles into the Cytosol
CHAPTER 10
Techniques to Distinguish Apoptosis from Necroptosis
INTRODUCTION
Techniques to Distinguish Apoptosis from
Necroptosis
PROTOCOLS
1 Crystal
Violet Assay for Determining Viability of Cultured Cells
2 Analysis
of Apoptosis and Necroptosis by Fluorescence-Activated Cell Sorting
3 Time-Lapse
Imaging of Cell Death
4 Ripoptosome
Analysis by Caspase-8 Coimmunoprecipitation
CHAPTER 11 Techniques
for the Detection of Autophagy in Primary Mammalian Cells
INTRODUCTION
Techniques for the Detection of Autophagy in
Primary Mammalian Cells
PROTOCOLS
1 Monitoring
Autophagic Flux by Using Lysosomal Inhibitors and Western Blotting of
Endogenous MAPILC3B
2 Monitoring
the Localization of MAPILC3B by Indirect Immunofluorescence
3 Analyzing
the Colocalization of MAPILC3 and Lysosomal Markers in Primary Cells
4 Detection
of p62 on Paraffin Sections by Immunohistochemistry
5 Detection
of Mitochondrial Mass, Damage, and Reactive Oxygen Species by Flow Cytometry
CHAPTER 12
Measuring Apoptosis in Mammals In Vivo
INTRODUCTION
Measuring Apoptosis in Mammals In Vivo
PROTOCOLS
1 Fluorodeoxyglucose-Based
Positron Emission Tomography Imaging to Monitor Drug Responses in Hematological
Tumors
2 Fluorodeoxyglucose-Based
Positron Emission Tomography Imaging to Monitor Drug Responses in Solid Tumors
3 Detection
of Apoptotic Cells Using Immunohistochemistry
4 Detection
of Apoptotic Cells Using Propidium Iodide Staining
CHAPTER 13 Studying
Apoptosis in Drosophila
INTRODUCTION
Studying Apoptosis in Drosophila
PROTOCOLS
1 Terminal
Deoxynucleotidy1 Transferase (TdT)-Mediated dUTP Nick-End Labeling (TUNEL) for
Detection of Apoptotic Cells in Drosophila
2 Using the
Vital Dye Acridine Orange to Detect Dying Cells in Drosophila
3
Immunostaining Using an Antibody against Active Caspase-3 to Detect
Apoptotic Cells in Drosophila
4 Analyzing
the Response of RNAi-Treated Drosophila Cells to Death Stimuli by Quantitative
Real-Time Polymerase Chain Reaction
5 Using
Synthetic Peptide Substrates to Measure Drosophila Caspase Activity
CHAPTER 14 Monitoring
Autophagy in Drosophila
INTRODUCTION
Monitoring Autophagy in Drosophila
PROTOCOLS
1 Lyso Tracker
Staining to Aid in Monitoring Autophagy in Drosophila
2 Monitoring
Autophagic Flux Using Ref(2)P, the Drosophila p62 Ortholog
3 Monitoring
Autophagy in Drosophila Using Fluorescent Reporters in the UAS-GAL4 System
4 Genetic
Manipulation of Autophagy in the Drosophila Ovary
CHAPTER 15 Analysis
of Apoptosis in Caenorhabditis elegans
INTRODUCTION
Analysis of Apoptosis in Caenorhabditis elegans
PROTOCOLS
1 Induction
of Germline Apoptosis in Caenorhabditis elegans
2
Visualizing Apoptosis in Embryos and the Germline of Caenorhabditis
elegans
3
Fluorescent Visualization of Germline Apoptosis in Living Caenorhabditis
elegans
4
High-Throughput RNAi Screening for Germline Apoptosis Genes in
Caenorhabditis elegans
5
Immunostaining for Markers of Apoptosis in the Caenorhabditis elegans
Germline
CHAPTER 16 Detection
of Autophagy in Caenorhabditis elegans
INTRODUCTION
Detection of Autophagy in Caenorhabditis elegans
PROTOCOLS
1 Detection
of Autophagy in Caenorhabditis elegans Using GFP::LGG-1 as an Autophagy Marker
2 Detecting
Autophagy in Caenorhabditis elegans Embryos Using Markers of P Granule
Degradation
3 Detection
of Autophagy in Caenorhabditis elegans by Western Blotting Analysis of LGG-1
4
RNAi-Mediated Inactivation of Autophagy Genes in Caenorhabditis elegans
CHAPTER 17 High-Throughput
Approaches to Measuring Cell Death
INTRODUCTION
High-Throughput Approaches to Measuring Cell Death
PROTOCOL
APPENDIX General
Safety and Hazardous Material Information
INDEX
