Books  Data Analysis & Modelling  Laboratory Practice 

Cell Death Techniques: A Laboratory Manual

Handbook / Manual

By: Ricky W Johnstone (Editor), John Silke (Editor)

514 pages, 54 colour & 73 b/w illustrations

Cold Spring Harbor Laboratory Press

Paperback | Sep 2015 | #223039 | ISBN-13: 9781621820055
Availability: Usually dispatched within 6 days Details
NHBS Price: £59.99 $76/€71 approx
Hardback | Sep 2015 | #223040 | ISBN-13: 9781621820123
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About this book

Regulated cell death, which is involved in nearly every aspect of animal development and physiology, can be challenging to study in the laboratory. There are multiple types of cell death, several interconnected signaling pathways can be activated, and the postmortem consequences may vary. Therefore, a combination of specialized techniques is often required to characterize and confirm cell death in the system of interest.

This laboratory manual provides a comprehensive suite of step-by-step protocols for inducing, detecting, visualizing, characterizing, and quantifying cell death in a variety of systems. The contributors describe strategies for identifying and evaluating the biochemical and morphological changes that occur during apoptosis, autophagy, necrosis, and other forms of cell death, as well as for pinpointing the genes and proteins that may be involved. The protocols employ flow cytometry, imaging and microscopy, RNAi, western blotting, enzymatic activity measurements, and other tools to assess phenomena such as DNA fragmentation, lysosomal membrane permeabilization, autophagic flux, apoptosome or ripoptosome formation, caspase activation, and cytochrome c release. Techniques are described for both in vitro systems and in vivo models, including mice, C. elegans, and Drosophila.

The authors also provide guidance on interpreting and presenting the results of cell death experiments, as well as advice on complementary procedures that may be required to confirm the results of a given experiment. This indispensable laboratory resource will enable all cell and developmental biologists, from the graduate level upward, to confidently carry out and comprehend a wide array of cell viability assays.


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
    John Silke and Ricky W. Johnstone
CHAPTER 2
Presentation and Interpretation of Figures in Cell Death Research Publications
INTRODUCTION
Presentation and Interpretation of Figures in Cell Death Research Publications
    David L. Vaux
PROTOCOLS
1 Preparing Figures from Cell Death Experimental Data for Publication
    David L. Vaux
2 Interpreting Cell Death Data from Figures in Published Papers
    David L. Vaux
CHAPTER 3
Dead Cert: Measuring Cell Death
INTRODUCTION
Dead Cert: Measuring Cell Death
    Lisa C. Crowley, Brooke J. Marfell, Adrian P. Scott, Jeanne A. Boughaba, Grace Chojnowski, Melinda E. Christensen, and Nigel J. Waterhouse
PROTOCOLS
1 Triggering Apoptosis in Hematopoietic Cells with Cytotoxic Drugs
    Lisa C. Crowley, Brooke J. Marfell, Adrian P. Scott, and Nigel J. Waterhouse
2 Triggering Death of Adherent Cells with Ultraviolet Radiation
    Lisa C. Crowley and Nigel J. Waterhouse
3 Measuring Cell Death by Trypan Blue Uptake and Light Microscopy
    Lisa C. Crowley, Brooke J. Marfell, Melinda E. Christensen, and Nigel J. Waterhouse
4 Measuring Cell Death by Propidium Iodide Uptake and Flow Cytometry
    Lisa C. Crowley, Adrian P. Scott, Brooke J. Marfell, Jeanne A. Boughaba, Grace Chojnowski, and Nigel J. Waterhouse
5 Measuring Survival of Adherent Cells with the Colony-Forming Assay
    Lisa C. Crowley, Melinda E. Christensen, and Nigel J. Waterhouse
6 Measuring Survival of Hematopoietic Cancer Cells with the Colony-Forming Assay in Soft Agar
    Lisa C. Crowley and Nigel J. Waterhouse
7 Morphological Analysis of Cell Death by Cytospinning Followed by Rapid Staining
    Lisa C. Crowley, Brooke J. Marfell, and Nigel J. Waterhouse
8 Analyzing Cell Death by Nuclear Staining with Hoechst 33342
    Lisa C. Crowley, Brooke J. Marfell, and Nigel J. Waterhouse
9 Detection of DNA Fragmentation in Apoptotic Cells byTUNEL
    Lisa C. Crowley, Brooke J. Marfell, and Nigel J. Waterhouse
10 Measuring the DNA Content of Cells in Apoptosis and at Different Cell-Cycle Stages by Propidium Iodide Staining and Flow Cytometry
    Lisa C. Crowley, Grace Chojnowski, and Nigel J. Waterhouse
11 Quantitation of Apoptosis and Necrosis by Annexin V Binding, Propidium Iodide Uptake, and Flow Cytometry
    Lisa C. Crowley, Brooke J. Marfell, Adrian P. Scott, and Nigel J. Waterhouse
12 Detecting Cleaved Caspase-3 in Apoptotic Cells by Flow Cytometry
    Lisa C. Crowley and Nigel J. Waterhouse
13 Analysis of Cytochrome c Release by Immunocytochemistry
    Lisa C. Crowley, Brooke J. Marfell, Adrian P. Scott, and Nigel J. Waterhouse
14 Measuring Mitochondrial Transmembrane Potential by TMRE Staining
    Lisa C. Crowley, Melinda E. Christensen, and Nigel J. Waterhouse
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
    Claudia Langlais, Michelle A. Hughes, Kelvin Cain, and Marion MacFarlane
PROTOCOLS
1 In Vitro Assembly and Analysis of the Apoptosome Complex
    Claudia Langlais, Michelle A. Hughes, Kelvin Cain, and Marion MacFarlane
2 Activation, Isolation, and Analysis of the Death-Inducing Signaling Complex
    Michelle A. Hughes, Claudia Langlais, Kelvin Cain, and Marion MacFarlane
CHAPTER 5
Biochemical Assays of the Proapoptotic Proteins Bak and Bax
INTRODUCTION
Biochemical Assays of the Proapoptotic Proteins Bak and Bax
    Grant Dewson
PROTOCOLS
1 Investigating Bax Subcellular Localization and Membrane Integration
    Grant Dewson
2 Investigating Bak/Bax Activating Conformation Change by Immunoprecipitation
    Grant Dewson
3 Detection of Bak/Bax Activating Conformation Change by Intracellular Flow Cytometry
    Grant Dewson
4 Investigating the Oligomerization of Bak and Bax during Apoptosis by Cysteine Linkage
    Grant Dewson
5 Blue Native PAGE and Antibody Gel Shift to Assess Bak and Bax Conformation Change and Oligomerization
    Grant Dewson
CHAPTER 6
Imaging-Based Methods for Assessing Caspase Activity in Single Cells
INTRODUCTION
Imaging-Based Methods for Assessing Caspase Activity in Single Cells
    Melissa J. Parsons, Markus Rehm, and Lisa Bouchier-Hayes
PROTOCOLS
1 Measuring Initiator Caspase Activation by Bimolecular Fluorescence Complementation
    Melissa J. Parsons and Lisa Bouchier-Hayes
2 Measuring Caspase Activity by Forster Resonance Energy Transfer
    Markus Rehm, Melissa J. Parsons, and Lisa Bouchier-Hayes
CHAPTER 7
Measuring Apoptosis: Caspase Inhibitors and Activity Assays
INTRODUCTION
Measuring Apoptosis: Caspase Inhibitors and Activity Assays
    Gavin P. McStay and Douglas R. Green
PROTOCOLS
1 Preparation of Cytosolic Extracts and Activation of Caspases by Cytochrome c
    Gavin P. McStay and Douglas R. Green
2 Assaying Caspase Activity In Vitro
    Gavin P. McStay and Douglas R. Green
3 Detection of Caspase Activity by Using Antibody-Based Techniques
    Gavin P. McStay and Douglas R. Green
4 Identification of Active Caspases by Using Affinity-Based Probes
    Gavin P. McStay and Douglas R. Green
5 Verification of a Putative Caspase Substrate
    Gavin P. McStay and Douglas R. Green
CHAPTER 8
Methods for Probing Lysosomal Membrane Permeabilization
INTRODUCTION
Methods for Probing Lysosomal Membrane Permeabilization
    Marja Jaattela and Jesper Nylandsted
PROTOCOLS
1 Quantification of Lysosomal Membrane Permeabilization by Cytosolic Cathepsin and b-N-Acetyl-Glucosaminidase Activity Measurements
    Marja Jaattela and Jesper Nylandsted
2 Visualizing Lysosomal Membrane Permeabilization by Fluorescent Dextran Release
    Anne-Marie Ellegaard, Marja Jaattela, and Jesper Nylandsted
3 A Method to Monitor Lysosomal Membrane Permeabilization by Immunocytochemistry
    Line Groth-Pedersen, Marja Jaattela, and Jesper Nylandsted
CHAPTER 9
Strategies for Assaying Lysosomal Membrane Permeabilization
INTRODUCTION
Strategies for Assaying Lysosomal Membrane Permeabilization
    Urska Repnik, Marusa Hafner Cesen, and Boris Turk
PROTOCOLS
1 The Use of Lysosomotropic Dyes to Exclude Lysosomal Membrane Permeabilization
    Urska Repnik, Marusa Hafner Cesen, and Boris Turk
2 Measuring Cysteine Cathepsin Activity to Detect Lysosomal Membrane Permeabilization
    Urska Repnik, Marusa Hafner Cesen, and Boris Turk
3 Studying Lysosomal Membrane Permeabilization by Analyzing the Release of Preloaded BSA-Gold Particles into the Cytosol
    Urska Repnik, Marusa Hafner Cesen, and Boris Turk
CHAPTER 10
Techniques to Distinguish Apoptosis from Necroptosis
INTRODUCTION
Techniques to Distinguish Apoptosis from Necroptosis
    Maria Feoktistova, Fredrik Wallberg, Tencho Tenev, Peter Geserick, Martin Leverkus, and Pascal Meier
PROTOCOLS
1 Crystal Violet Assay for Determining Viability of Cultured Cells
    Maria Feoktistova, Peter Geserick, and Martin Leverkus
2 Analysis of Apoptosis and Necroptosis by Fluorescence-Activated Cell Sorting
    Fredrik Wallberg, Tencho Tenev, and Pascal Meier
3 Time-Lapse Imaging of Cell Death Fredrik Wallberg, Tencho Tenev, and Pascal Meier
4 Ripoptosome Analysis by Caspase-8 Coimmunoprecipitation
    Maria Feoktistova, Peter Geserick, and Martin Leverkus
CHAPTER 11
Techniques for the Detection of Autophagy in Primary Mammalian Cells
INTRODUCTION
Techniques for the Detection of Autophagy in Primary Mammalian Cells
    Daniel Puleston, Kanchan Phadwal, Alexander Scarth Watson, Elizabeth J Soilleux, Suganthi Chittaranjan, Svetlana Bortnik, Sharon M. Gorski, Nicholas Ktistakis, and Anna Katharina Simon
PROTOCOLS
1 Monitoring Autophagic Flux by Using Lysosomal Inhibitors and Western Blotting of Endogenous MAP1LC3B
    Suganthi Chittaranjan, Svetlana Bortnik, and Sharon M. Gorski
2 Monitoring the Localization of MAP1LC3B by Indirect Immunofluorescence
    Nicholas T. Ktistakis
3 Analyzing the Colocalization of MAP1LC3 and Lysosomal Markers in Primary Cells
    Kanchan Phadwal
4 Detection of p62 on Paraffin Sections by Immunohistochemistry
    Alexander S. Watson and Elizabeth J. Soilleux
5 Detection of Mitochondrial Mass, Damage, and Reactive Oxygen Species by Flow Cytometry
    Daniel Puleston
CHAPTER 12
Measuring Apoptosis in Mammals In Vivo
INTRODUCTION
Measuring Apoptosis in Mammals In Vivo
    Andrea Newbold, Ben P. Martin, Carleen Cullinane, and Michael Bots
PROTOCOLS
1 Fluorodeoxyglucose-Based Positron Emission Tomography Imaging to Monitor Drug Responses in Hematological Tumors
    Andrea Newbold, Ben P. Martin, Carleen Cullinane, and Michael Bots
2 Fluorodeoxyglucose-Based Positron Emission Tomography Imaging to Monitor Drug Responses in Solid Tumors
    Andrea Newbold, Ben P. Martin, Carleen Cullinane, and Michael Bots
3 Detection of Apoptotic Cells Using Immunohistochemistry
    Andrea Newbold, Ben P. Martin, Carleen Cullinane, and Michael Bots
4 Detection of Apoptotic Cells Using Propidium Iodide Staining
    Andrea Newbold, Ben P. Martin, Carleen Cullinane, and Michael Bots
CHAPTER 13
Studying Apoptosis in Drosophila
INTRODUCTION
Studying Apoptosis in Drosophila
    Donna Denton and Sharad Kumar
PROTOCOLS
1 Terminal Deoxynucleotidyl Transferase (TdT)-Mediated dUTP Nick-End Labeling (TUNEL) for Detection of Apoptotic Cells in Drosophila
    Donna Denton and Sharad Kumar
2 Using the Vital Dye Acridine Orange to Detect Dying Cells in Drosophila
    Donna Denton and Sharad Kumar
3 Immunostaining Using an Antibody against Active Caspase-3 to Detect Apoptotic Cells in Drosophila
    Donna Denton and Sharad Kumar
4 Analyzing the Response of RNAi-Treated Drosophila Cells to Death Stimuli by Quantitative Real-Time Polymerase Chain Reaction
    Donna Denton and Sharad Kumar
5 Using Synthetic Peptide Substrates to Measure Drosophila Caspase Activity
    Donna Denton and Sharad Kumar
CHAPTER 14
Monitoring Autophagy in Drosophila
INTRODUCTION
Monitoring Autophagy in Drosophila
    Lindsay DeVorkin and Sharon M. Gorski
PROTOCOLS
1 LysoTracker Staining to Aid in Monitoring Autophagy in Drosophila
    Lindsay DeVorkin and Sharon M. Gorski
2 Monitoring Autophagic Flux Using Ref(2)P, the Drosophila p62 Ortholog
    Lindsay DeVorkin and Sharon M. Gorski
3 Monitoring Autophagy in Drosophila Using Fluorescent Reporters in the UAS-GAL4 System
    Lindsay DeVorkin and Sharon M. Gorski
4 Genetic Manipulation of Autophagy in the Drosophila Ovary
    Lindsay DeVorkin and Sharon M. Gorski
CHAPTER 15
Analysis of Apoptosis in Caenorhabditis elegans
INTRODUCTION
Analysis of Apoptosis in Caenorhabditis elegans
    Benjamin Lant and W. Brent Derry
PROTOCOLS
1 Induction of Germline Apoptosis in Caenorhabditis elegans
    Benjamin Lant and W. Brent Derry
2 Visualizing Apoptosis in Embryos and the Germline of Caenorhabditis elegans
    Benjamin Lant and W. Brent Derry
3 Fluorescent Visualization of Germline Apoptosis in Living Caenorhabditis elegans
    Benjamin Lant and W. Brent Derry
4 High-Throughput RNAi Screening for Germline Apoptosis Genes in Caenorhabditis elegans
    Benjamin Lant and W. Brent Derry
5 Immunostaining for Markers of Apoptosis in the Caenorhabditis elegans Germline
    Benjamin Lant and W. Brent Derry
CHAPTER 16
Detection of Autophagy in Caenorhabditis elegans
INTRODUCTION
Detection of Autophagy in Caenorhabditis elegans
    Nicholas J. Palmisano and Alicia Meléndez
PROTOCOLS
1 Detection of Autophagy in Caenorhabditis elegans Using GFP::LGG-1 as an Autophagy Marker
    Nicholas J. Palmisano and Alicia Meléndez
2 Detecting Autophagy in Caenorhabditis elegans Embryos Using Markers of P Granule Degradation
    Nicholas J. Palmisano and Alicia Meléndez
3 Detection of Autophagy in Caenorhabditis elegans by Western Blotting Analysis of LGG-1
    Nicholas J. Palmisano and Alicia Meléndez
4 RNAi-Mediated Inactivation of Autophagy Genes in Caenorhabditis elegans
    Nicholas J. Palmisano and Alicia Meléndez
CHAPTER 17
High-Throughput Approaches to Measuring Cell Death
INTRODUCTION
High-Throughput Approaches to Measuring Cell Death
    Darren N. Saunders, Katrina J. Falkenberg, and Kaylene J. Simpson
PROTOCOL
1 A High-Throughput, Multiplex Cell Death Assay Using an RNAi Screening Approach
    Katrina J. Falkenberg, Darren N. Saunders, and Kaylene J. Simpson
APPENDIX
General Safety and Hazardous Material Information
INDEX


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