Apoptosis A Practical Approach

by Studzinski, George P.

• ISBN13: 9780199637850
• ISBN10: 0199637857
• Format: Paperback
• Copyright: 2000-03-09
• Publisher: Oxford University Press

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