Dna Fragmentation Filter Elution Assay

List of Contributors xv

Abbreviations xix

1. Overview of apoptosis 1

George P. Studzinski

1. General introduction and overview of contents 1

2. Historical perspective 5

3. Distinction of apoptosis from other forms of cell death 5

4. Apoptotic cascades 8

5. Time course of apoptotic cascades 8

6. Selection of methods 11

Procedure for determination of increased mitochondrial to nuclear

DNA ratio, for the detection and quantitation of apoptosis 12

7. Pitfalls 14 References 16

2. Morphological recognition of apoptotic cells 19

James W. Wilson and Christopher S. Potten

1. Introduction 19

Key morphological features of apoptotic cells 19

2. Light and fluorescent microscopy techniques for the assessment of apoptosis 20

Preparation of cell or tissue samples 20

Nuclear counterstains 23

3. Electron microscopic techniques 27

4. Quantitation of apoptotic events 29

Methods 29

Problems in scoring apoptotic events 31

5. In situ detection of DNA strand breaks 33

6. Other techniques 37

7. Conclusions 38 References 38

3. Assessment of DNA damage in apoptosis 41

Akira Yoshida, Rong-Guang Shao, and Yves Pommier

1. Introduction 41

2. Types of DNA fragmentation 41

3. Measurement of high molecular weight DNA fragmentation by pulse-field gel electrophoresis 43

Pulse-field gel electrophoresis procedures 44

Strategies for electrophoretic separations 46

4. Detection of internucleosomal DNA fragmentation by standard agarose gel electrophoresis 47

5. Filter elution assay to measure apoptotic DNA fragmentation 48

Equipment 49

Filters and solutions 49

DNA labelling and preparation of experimental cell cultures 50

Protocols 51

Counting samples and computations 52

References 53

4. Analysis of cell death by flow and laser-scanning cytometry 57

Zbigniew Darzynkiewicz, Elzbieta Bedner, and Xun Li

1. Introduction 57

2. Preparation of cells for analysis by LSC. Detection of apoptotic cells based on changes in nuclear chromatin 59

3. Light-scattering properties of apoptotic and necrotic cells 61

4. Analysis of mitochondrial transmembrane potential (Ai|>m) 63

5. Detection of apoptotic cells exposing phosphatidylserine on plasma membrane 67

6. Detection of apoptotic cells based on their fractional DNA content 68

7. Identification of apoptotic cells based on the presence of

DNA strand breaks 71

8. Identification of apoptotic cells based on the increased

DNA sensitivity to denaturation 75

9. General comments 77 References 78

5. Cell-mediated cytotoxicity and cell death receptors 8i

Javier Naval and Alberto Anel

1. Overview of pathways for cell-mediated cytotoxicity 81

Fas-based cytotoxicity 82

Perforin/granzyme-based cytotoxicity 83

2. Standard methods for the evaluation of cell-mediated cytotoxicity 84

Chromium release assay 84

[125I]Iododeoxyuridine (125IUdR) release assay 87

The JAM test 89

BLT-esterase release assay 91 Estimation of target cell nuclear fragmentation using fluorescent dyes 92

Activation-induced cell death (AICD) 94

3. Separate studies of Fas- and perforin/granzyme-based cytotoxicity 95

Fas-based cytotoxicity in the absence of perforin/granzyme contribution 95 Perforin/granzyme-based cytotoxicity in the absence of FasL

contribution 96

4. Use of caspase and granzyme inhibitors in cell-mediated cytotoxicity assays 97

Caspase inhibitors 97

Granzyme inhibitors 99

References 100

6. Sphingolipids as messengers of cell death 105

Gary M. Jenkins and YusufA. Hannun

1. Introduction 105

2. Sphingolipids and their role in apoptosis 105

Overview of sphingolipids 105

Role of sphingolipids in apoptosis 106

Strategies and considerations in evaluating sphingolipids 107

3. Extraction of sphingolipids and normalization by lipid phosphate 108

Extraction of sphingosines, ceramides, and sphingomyelins 108

Alkaline hydrolysis of sphingolipids 111 Lipid phosphate measurement for normalization of experimental samples 112

4. Analysis of sphingoid backbones 113

HPLC on sphingoid backbones 113

5. Analysis of ceramides 115

6. Analysis of sphingomyelins 118

7. Measurement of SMase and SM synthase activities 120 References 123

7. Cytochemical detection of cytoskeletal and nucleoskeletal changes during apoptosis 125

Manon van Engeland, Bert Schutte, Anton H. N. Hopman, Frans C. S. Ramaekers, and Chris P. M. Reutelingsperger

1. Introduction 125

2. Cyto- and nucleoskeletal changes during apoptosis 126

General 126

Microfilaments and microfilament-associated proteins 126

Microtubules 126

Intermediate filaments 127

Nucleoskeletal components 128 Generation of neo-epitopes in cytoskeletal proteins during apoptosis 128

3. Apoptosis detection systems 129

Overview 129

The annexin V affinity assay 129

Detection of apoptosis using the TUNEL assay 135

References 138

8. Metabolic alterations associated with apoptosis 141

Ana P. Costa-Pereira and Thomas G. Cotter

1. Introduction 141

2. Detection of changes in the mitochondrial transmembrane potential 142

3. Detection of intracellular reactive oxygen intermediates

4. Determination of glutathione levels and its oxidative state 146

5. Measurement of catalase in cells undergoing apoptosis 153 References 155

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