Technical guide to Cell Proliferation and Apoptosis Methods (Roche) (archive)
This is a technical guide to Cell Proliferation and Apoptosis Methods (Roche).
Guide to Cell Proliferation and Apoptosis Methods
Table of Contents
Chapter 1: Cell Death - Apoptosis and Necrosis
1.1 Introduction 2
1.1.1 Terminology of cell death 2
1.1.2 Differences between necrosis and apoptosis 3
1.1.3 Apoptotic Pathways 4
1.2 Apoptosis Assay Methods 6
Method/Product selection guide 7
1.2.1 Methods for studying apoptosis in cell populations 8
1.2.1.1 Assays that measure DNA fragmentation 8
Apoptotic DNA Ladder Kit 11
Cell Death Detection ELISAPLUS 13
1.2.1.2 Assays that measure apoptosis-induced proteases (caspase) 16
Caspase 3 Activity Assay 17
Anti-PARP 20
1.2.1.3 Summary of methods for studying apoptosis in cell populations 22
1.2.2 Methods for studying apoptosis in individual cells 24
1.2.2.1 The TUNEL enzymatic labeling assay 24
In Situ Cell Death Detection Kit, Fluorescein 27
In Situ Cell Death Detection Kit, AP 29
In Situ Cell Death Detection Kit, POD 29
1.2.2.2 Assays that measure membrane alterations 31
Annexin-V-FLUOS 32
Annexin-V-FLUOS Staining Kit 32
Annexin-V-AlexaTM 568 32
Annexin-V-Biotin 34
1.2.2.3 Assays that use DNA stains 36
DAPI, Ethidium bromide, Propidium iodide 36
1.2.2.4 Summary of methods for studying apoptosis in individual cells 38
1.2.3 Detection of apoptosis-related proteins 40
Anti-Fas (CD95/Apo-1) 41
Anti-Fas-Biotin (CD95/Apo-1) 42
Anti-Bcl-2 oncoprotein, human 44
Anti-p53 Protein, mutant 46
Anti-p53 Protein pan 46
Anti-p53 pan 46
Anti-p53, Biotin labeled 46
Anti-p53, Peroxidase labeled 46
p53pan ELISA 48
1.3 Cytotoxicity Assay Methods 50
1.3.1 Relationship between cytotoxicity, apoptosis, and necrosis 50
1.3.2 Methods for studying cytotoxicity 50
1.3.2.1 Assays the measure plasma membrane leakage 51
Cytotoxicity Detection Kit (LDH) 52
Cellular DNA Fragmentation ELISA 54
1.3.2.2 Assays that measure metabolic activity 58
Cell Proliferation Kit I (MTT) 58
Cell Proliferation Kit II (XTT) 58
Cell Proliferation Reagent WST-1 58
1.3.2.3 Summary of methods for studying cytotoxicity 60
Chapter 2: Cell Proliferation and Viability
2.1 Introduction 64
2.1.1 Terminology of cell proliferation and viability 64
2.1.2 Cell cycle 64
2.2 Cell proliferation/viability assay methods 67
Method/Product selection guide 68
2.2.1 Methods for studying cell proliferation and viability in cell populations 70
2.2.1.1 Assays that measure metabolic activity 70
Cell Proliferation Kit I (MTT) 73
Cell Proliferation Kit II (XTT) 74
Cell Proliferation Reagent WST-1 75
2.2.1.2 Assays that measure DNA synthesis 77
BrdU Labeling and Detection Kit III 79
Cell Proliferation ELISA, BrdU (colorimetric) 81
Cell Proliferation ELISA, BrdU (chemiluminescence) 81
2.2.1.3 Summary of methods for studying cell proliferation and cell viability in cell populations 84
2.2.1.4 Single reagents for the measurement of DNA synthesis 84
2.2.2 Methods for studying cell proliferation and viability in individual cells 86
2.2.2.1 Assays that measure DNA synthesis 86
BrdU Labeling and Detection Kit I 87
BrdU Labeling and Detection Kit II 87
BrdU Labeling and Detection Kit, FLUOS 88
In situ Cell Proliferation Kit, FLUOS 90
In Situ Cell Proliferation Kit, AP 93
Anti-BrdU, formalin grade 93
Anti-BrdU-Fluorescein 93
Anti-BrdU-AP, F(ab)2 fragments 93
Anti-BrdU-Peroxidase, Fab fragment 93
2.2.2.2 Assays that monitor expression of cell cycle-associated antigens 96
Monoclonal antibodies to cell cycle-associated antigens 97
2.2.2.3 Summary of methods for studying cell proliferation and viability in individual cells 100
Chapter 3: Appendix
3.1 Technical tips 104
3.1.1 Selected frequently asked questions (FAQs) about cell death assays 104
3.1.2 Technical tips on the TUNEL method 105
3.1.2.1 TUNEL: Improvement and evaluation of the method for in situ apoptotic cell identification 105
3.1.2.2 TUNEL protocol for tissues which tend to give false positives 105
3.1.2.3 Tips for avoiding or eliminating potential TUNEL labeling artifacts 107
3.1.3 Technical tips on the use of Annexin-V-Biotin for light microscope detection 109
3.1.4 Technical tips on the use of the Apoptotic DNA Ladder Kit on tissue samples 109
3.1.5 Technical tips on the Cell Proliferation ELISA kits 110
3.2 Special applications of cell death and cell proliferation methods 111
3.2.1 TUNEL assays 111
3.2.1.1 Discrimination between dead and viable apoptotic cells using two-color TdT assay and surface labeling as detected by flow cytometry 111
3.2.1.2 The use of flow cytometry for concomitant detection of apoptosis and cell cycle analysis 111
3.2.1.3 Comparison of two cell death detection methods: In situ nick translation and TUNEL 112
3.2.1.4 Fixation of tissue sections for TUNEL combined with staining for thymic epithelial cell marker 112
3.2.2 Metabolic assays 113
3.2.2.1 Biochemical and cellular basis of cell proliferation assays that use tetrazolium salts 113
3.2.3 Annexin assays 113
3.2.3.1 The use of annexin for concomitant detection of apoptosis and cellular phenotype 113
3.2.4 BrdU assays 114
3.2.4.1 Detection of bromodeoxyuridine in paraffin-embedded tissue sections using microwave antigen retrieval is dependent on the mode of tissue fixation 114
3.3 References 115
3.3.1 Apotosis- related parameters - Abbreviations and References 115
3.3.2 Examples for applications for Boehringer Mannheim products 120
3.3.3 General references 127
3.4 General abbreviations 129
3.5 Ordering Guide 131
3.6 Index 134
Contributors: Andrew Wyllie, Ph.D.
Vicki Donahue, M.S.
Bertram Fischer, Ph.D.
David Hill, Ph.D.
Joe Keesey, Ph.D.
Simone Manzow, Ph.D.
Editorial Management Doris Eisel.
Georg Fertig, Ph.D
Bertram Fischer, Ph.D.
Simone Manzow, Ph.D
Karl Schmelig
Last update 28-Jan-2005, Rating Excellent! of 6 votes.
