9780199637850

Apoptosis A Practical Approach

by
  • ISBN13:

    9780199637850

  • ISBN10:

    0199637857

  • Edition: 1st
  • Format: Paperback
  • Copyright: 2000-03-09
  • Publisher: Oxford University Press

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Summary

This text is designed to provide conceptual outlines and detailed procedures for basic and advanced studies of cell death by apoptosis. Chapters on the recognition of apoptosis as distinguished from necrosis and nonspecific cell DNA damage, are followed by a systematic examination of theestablished and the principal novel methodologies utilized by some leading laboratories conducting research on apoptosis. The organization is on the lines of signalling for apoptosis, the apoptotic cascade, and the execution of apoptosis. A wide variety of procedures are provided which will enablethe reader to participate in cutting-edge research.

Table of Contents

List of Contributors
xv
Abbreviations xix
Overview of apoptosis
1(18)
George P. Studzinski
General introduction and overview of contents
1(4)
Historical perspective
5(1)
Distinction of apoptosis from other forms of cell death
5(3)
Apoptotic cascades
8(1)
Time course of apoptotic cascades
8(3)
Selection of methods
11(3)
Procedure for determination of increased mitochondrial to nuclear DNA ratio, for the detection and quantitation of apoptosis
12(2)
Pitfalls
14(5)
References
16(3)
Morphological recognition of apoptotic cells
19(22)
James W. Wilson
Christopher S. Potten
Introduction
19(1)
Key morphological features of apoptotic cells
19(1)
Light and fluorescent microscopy techniques for the assessment of apoptosis
20(7)
Preparation of cell or tissue samples
20(3)
Nuclear counterstains
23(4)
Electron microscopic techniques
27(2)
Quantitation of apoptotic events
29(4)
Methods
29(2)
Problems in scoring apoptotic events
31(2)
In situ detection of DNA strand breaks
33(4)
Other techniques
37(1)
Conclusions
38(3)
References
38(3)
Assessment of DNA damage in apoptosis
41(16)
Akira Yoshida
Rong-Guang Shao
Yves Pommier
Introduction
41(1)
Types of DNA fragmentation
41(2)
Measurement of high molecular weight DNA fragmentation by pulse-field gel electrophoresis
43(4)
Pulse-field gel electrophoresis procedures
44(2)
Strategies for electrophoretic separations
46(1)
Detection of internucleosomal DNA fragmentation by standard agarose gel electrophoresis
47(1)
Filter elution assay to measure apoptotic DNA fragmentation
48(9)
Equipment
49(1)
Filters and solutions
49(1)
DNA labelling and preparation of experimental cell cultures
50(1)
Protocols
51(1)
Counting samples and computations
52(1)
References
53(4)
Analysis of cell death by flow and laser-scanning cytometry
57(24)
Zbigniew Darzynkiewicz
Elzbieta Bedner
Xun Li
Introduction
57(2)
Preparation of cells for analysis by LSC. Detection of apoptotic cells based on changes in nuclear chromatin
59(2)
Light-scattering properties of apoptotic and necrotic cells
61(2)
Analysis of mitochondrial transmembrane potential (Δψm)
63(4)
Detection of apoptotic cells exposing phosphatidylserine on plasma membrane
67(1)
Detection of apoptotic cells based on their fractional DNA content
68(3)
Identification of apoptotic cells based on the presence of DNA strand breaks
71(4)
Identification of apoptotic cells based on the increased DNA sensitivity to denaturation
75(2)
General comments
77(4)
References
78(3)
Cell-mediated cytotoxicity and cell death receptors
81(24)
Javier Naval
Alberto Anel
Overview of pathways for cell-mediated cytotoxicity
81(3)
Fas-based cytotoxicity
82(1)
Perforin/granzyme-based cytotoxicity
83(1)
Standard methods for the evaluation of cell-mediated cytotoxicity
84(11)
Chromium release assay
84(3)
[125I]Iododeoxyuridine (125IUdR) release assay
87(2)
The JAM test
89(2)
BLT-esterase release assay
91(1)
Estimation of target cell nuclear fragmentation using fluorescent dyes
92(2)
Activation-induced cell death (AICD)
94(1)
Separate studies of Fas- and perforin/granzyme-based cytotoxicity
95(2)
Fas-based cytotoxicity in the absence of perforin/granzyme contribution
95(1)
Perforin/granzyme-based cytotoxicity in the absence of FasL contribution
96(1)
Use of caspase and granzyme inhibitors in cell-mediated cytotoxicity assays
97(8)
Caspase inhibitors
97(2)
Granzyme inhibitors
99(1)
References
100(5)
Sphingolipids as messengers of cell death
105(20)
Gary M. Jenkins
Yusuf A. Hannun
Introduction
105(1)
Sphingolipids and their role in apoptosis
105(3)
Overview of sphingolipids
105(1)
Role of sphingolipids in apoptosis
106(1)
Strategies and considerations in evaluating sphingolipids
107(1)
Extraction of sphingolipids and normalization by lipid phosphate
108(5)
Extraction of sphingosines, ceramides, and sphingomyelins
108(3)
Alkaline hydrolysis of sphingolipids
111(1)
Lipid phosphate measurement for normalization of experimental samples
112(1)
Analysis of sphingoid backbones
113(2)
HPLC on sphingoid backbones
113(2)
Analysis of ceramides
115(3)
Analysis of sphingomyelins
118(2)
Measurement of SMase and SM synthase activities
120(5)
References
123(2)
Cytochemical detection of cytoskeletal and nucleoskeletal changes during apoptosis
125(16)
Manon van Engeland
Bert Schutte
Anton H. N. Hopman
Frans C. S. Ramaekers
Chris P. M. Reutelingsperger
Introduction
125(1)
Cyto- and nucleoskeletal changes during apoptosis
126(3)
General
126(1)
Microfilaments and microfilament-associated proteins
126(1)
Microtubules
126(1)
Intermediate filaments
127(1)
Nucleoskeletal components
128(1)
Generation of neo-epitopes in cytoskeletal proteins during apoptosis
128(1)
Apoptosis detection systems
129(12)
Overview
129(1)
The annexin V affinity assay
129(6)
Detection of apoptosis using the TUNEL assay
135(3)
References
138(3)
Metabolic alterations associated with apoptosis
141(16)
Ana P. Costa-Pereira
Thomas G. Cotter
Introduction
141(1)
Detection of changes in the mitochondrial transmembrane potential
142(2)
Detection of intracellular reactive oxygen intermediates (ROI)
144(2)
Determination of glutathione levels and its oxidative state
146(7)
Measurement of catalase in cells undergoing apoptosis
153(4)
References
155(2)
Methods of measuring Bcl-2 family proteins and their functions
157(58)
John C. Reed
Zhihua Xie
Shinichi Kitada
Juan M. Zapata
Qunli Xu
Sharon Schendel
Maryla Krajewska
Stanislaw Krajewski
Introduction
157(1)
Immunoblot analysis
157(8)
Cell lysis and tissue processing procedures
158(3)
Immunoblotting procedure
161(4)
Detection of Bcl-2 family proteins by flow cytometry (FACS)
165(2)
Indirect immunofluorescence procedure
166(1)
Antibodies
167(1)
Northern blot analysis of BCL-2 mRNA
167(4)
General guidelines for RNA blotting
168(1)
Pre-hybridization and hybridization procedures
169(1)
Solutions
170(1)
Reverse transcriptase-polymerase chain reaction (RT-PCR)
171(4)
cDNA synthesis
172(1)
PCR amplication
172(1)
Southern blot analysis of PCR products
173(1)
Primers and probes for PCR
174(1)
Immunohistochemical detection of Bcl-2 family proteins in tissues
175(12)
Procedures for tissue preparation, embedding, and sectioning
177(3)
Pre-staining sample preparation
180(3)
Immunostaining
183(2)
Counterstaining and mounting
185(1)
Solutions
186(1)
Immunohistochemical detection of Bcl-2 protein in cultured cells
187(4)
Procedures
188(1)
Immunostaining
189(2)
Solutions
191(1)
Production of recombinant Bcl-2 family proteins in bacteria
191(10)
Human Bcl-2
192(2)
Human Bcl-XL protein
194(3)
Mouse Bax protein
197(2)
Human Bid protein
199(2)
Measurements of pore formation by Bcl-2 family proteins
201(4)
Liposome preparation
202(2)
Channel activity measurements
204(1)
Methods of assaying Bcl-2 and Bax family protein function in yeast
205(7)
Plasmid considerations
205(2)
Saccharomyces cerevisiae strains
207(1)
Media and growth conditions
207(2)
Transformation of S. cerevisiae by the LiOAc method
209(1)
Assay for Bax-induced lethality in S. cerevisiae
210(2)
Summary
212(3)
References
212(3)
Methods for detecting proteolysis during apoptosis in intact cells
215(24)
April L. Blajeski
Scott H. Kaufmann
Introduction
215(1)
Involvement of proteases in apoptosis
215(2)
Caspase nomenclature and classification
217(1)
Detection of procaspases and their cleavage products by immunoblotting
218(6)
Theoretical considerations
218(4)
Practical considerations in immunoblotting for caspases
222(1)
Sample preparation and immunoblotting
222(2)
Cleavage of caspase targets
224(2)
Establishing that proteases are activated in situ
224(1)
Establishing that a polypeptide is cleaved by caspases
224(2)
Assays of caspase activity
226(4)
Theoretical considerations of caspase activity assays
227(1)
Measuring caspase activity in cell lysates
227(3)
Detection of active caspases by affinity labelling
230(4)
Theoretical considerations in affinity labelling
230(2)
Practical considerations in the use of affinity labelling reagents
232(1)
Labelling and detecting enzymatically active caspases
232(2)
Studying the biological effects of caspase cleavages
234(5)
A molecular approach for analysis of the effects of substrate cleavage
235(1)
Use of caspase inhibitors: promises and pitfalls
235(1)
References
236(3)
List of Suppliers 239(4)
Index 243

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