Apoptosis and Cell Proliferation Roche Applied Science
 
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Table of contents
A Cell Death — Apoptosis and Necrosis Page
1. Introduction 2
1.1 Terminology of cell death 2
1.2 Differences between necrosis and apoptosis 3
1.3 Apoptotic Pathways 5
 
2 Apoptosis Assay Methods 7
Method/Product selection guide 8
2.1 Methods for studying apoptosis in cell populations 10
2.1.1 Assays that measure DNA fragmentation 10
 Apoptotic DNA Ladder Kit 13
 Cell Death Detection ELISAPLUS 15
2.1.2 Assays that measure apoptosis-induced proteases (caspases) 18
 M30 CytoDEATH 19
 M30 CytoDEATH, Fluorescein 19
 Caspase 3 Activity Assay 22
 Homogeneous Caspases Assay 25
 Anti-PARP 27
2.1.3 Summary of methods for studying apoptosis in cell populations 30
2.2 Methods for studying apoptosis in individual cells 32
2.2.1 The TUNEL enzymatic labeling assay 33
 In Situ Cell Death Detection Kit, Fluorescein 36
 In Situ Cell Death Detection Kit, TMR red 36
 In Situ Cell Death Detection Kit, AP 39
 In Situ Cell Death Detection Kit, POD 39
2.2.2 Assays that measure membrane alterations 43
 Annexin-V-FLUOS 44
 Annexin-V-FLUOS Staining Kit 44
 Annexin-V-Alexa 568 44
 Annexin-V-Biotin 47
2.2.3 Assays that use DNA stains 49
 DAPI, Propidium iodide 50
2.2.4 Summary of methods for studying apoptosis in individual cells 52
2.3 Detection of apoptosis-related proteins 54
 Anti-Fas (CD95/Apo-1) 55
 p53 pan ELISA 56
 
3 Cytotoxicity Assay Methods 58
3.1 Relationship between cytotoxicity, apoptosis and necrosis 58
3.2 Methods for studying cytotoxicity 59
3.2.1 Assays that measure plasma membrane leakage 59
 Cytotoxicity Detection Kit (LDH) 61
 Cellular DNA Fragmentation ELISA 64
3.2.2 Assays that measure metabolic activity 68
 Cell Proliferation Kit I (MTT) 68
 Cell Proliferation Kit II (XTT) 68
 Cell Proliferation Reagent WST-1 68
3.2.3 Summary of methods for studying cytotoxicity 70
 
B Cell Proliferation and Viability Page
1. Introduction 74
1.1 Terminology of cell proliferation and viability 74
1.2 Cell Cycle 75
 
2. Cell proliferation/viability assay methods 78
Method/Product selection guide 80
2.1 Methods for studying cell proliferation and viability in cell populations 82
2.1.1 Assays that measure metabolic activity 82
 Cell Proliferation Kit I (MTT) 85
 Cell Proliferation Kit II (XTT) 86
 Cell Proliferation Reagent WST-1 87
2.1.2 Assays that measure DNA synthesis 89
 5'-Bromo-2'-deoxy-uridine Labeling and Detection Kit III 91
 Cell Proliferation ELISA, BrdU (colorimetric) 94
 Cell Proliferation ELISA, BrdU (chemiluminescence) 94
2.1.3 Summary of methods for studying cell proliferation and cell viability in cell populations 98
2.1.4 Single reagents for the measurement of DNA synthesis 98
2.2 Methods for studying cell proliferation and viability in individual cells 100
2.2.1 Assays that measure DNA synthesis 100
 5'-Bromo-2'-deoxy-uridine Labeling and Detection Kit I 101
 5'-Bromo-2'-deoxy-uridine Labeling and Detection Kit II 101
 In Situ Cell Proliferation Kit, FLUOS 102
 Anti-BrdU, formalin grade 105
 Anti-BrdU-Fluorescein 105
 Anti-BrdU-Peroxidase, Fab fragment 105
2.2.2 Summary of methods for studying cell proliferation and viability in individual cells 108
 
C Appendix Page
1. Technical Tips 112
1.1 Selected frequently asked questions (FAQs) about cell death assays 112
1.2 Technical tips on the TUNEL method 113
1.2.1 TUNEL: Improvement and evaluation of the method for in situ apoptotic cell identification 113
1.2.2 TUNEL protocol for tissues which tend to give false positives 114
1.2.3 Tips for avoiding or eliminating potential TUNEL labeling artifacts 115
1.3 Technical tips on the use of Annexin-V-Biotin for light microscope detection 117
1.4 Technical tips on the use of the Apoptotic DNA Ladder Kit on tissue samples 118
1.5 Technical tips on the Cell Proliferation ELISA Kits 118
 
2. Special applications of cell death and cell proliferation methods 119
2.1 TUNEL assays 119
2.1.1 Discrimination between dead and viable apoptotic cells using two-color TdT assay and surface labeling as detected by flow cytometry 119
2.1.2 The use of flow cytometry for concomitant detection of apoptosis and cell cycle analysis 119
2.1.3 Comparison of two cell death detection methods: In situ nick translation and TUNEL 120
2.1.4 Fixation of tissue sections for TUNEL combined with staining for thymic epithelial cell marker 120
2.2 Metabolic assays 121
2.2.1 Biochemical and cellular basis of cell proliferation assays that use tetrazolium salts 121
2.3 Annexin assays 121
2.3.1 The use of annexin for concomitant detection of apoptosis and cellular phenotype 121
2.4 BrdU assays 122
2.4.1 Detection of bromodeoxyuridine in paraffin-embedded tissue sections using microwave antigen retrieval is dependent on the mode of tissue fixation 122
 
3. References 123
3.1 Apoptosis-related parameters – Abbreviations and References 123
3.2 Examples for applications of Roche Applied Science products 128
3.3 General references 154
 
4. General abbreviations 158
 
5. Ordering Guide 161
 
6. Index 164
 
 
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