| 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 |
