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This book has sold over 10,000 copies since its publication in 1986, and remains one of the most popular titles in the Practical Approach Series. This new, thoroughly updated edition details the recent progress made in this field. There have been significant advances in areas such as serum-free media, scale-up, and flow cytometry. As these techniques have developed as tools for cell biologists, their availability to the non-specialist has also increased dramatically. Use of the tetrazolium salt MTT as a colorimetric indicator of viability has made a considerable impact on cytotoxicity assay, and DNA fingerprinting has revolutionized the identification of individual cell strains. These and other developments in the techniques described have made this new edition essential. This cohesive volume presents techniques in a readily accessible form, and provides detailed protocols throughout. This volume will be of use to researchers involved in both biological research and the commercial exploitation of animal cell culture.
| List of contributors | ||
| Abbreviations | ||
| 1 | Introduction to basic principles | 1 |
| 2 | Biology of cells in culture | 1 |
| 3 | Choice of materials | 3 |
| 4 | Preparation | 9 |
| 2 | Towards serum-free, chemically defined media for mammalian cell culture | 15 |
| 2 | Role of serum in cell culture | 15 |
| 3 | Strategies for designing serum-free cultures and appropriate media | 21 |
| 4 | Practical suggestions for the preparation and handling of serum-free media | 38 |
| 5 | Procedures for coating of culture surface with biomatrix | 41 |
| 3 | Scaling-up of animal cell cultures | 47 |
| 2 | General methods and culture parameters | 48 |
| 3 | Monolayer culture | 62 |
| 4 | Suspension culture | 78 |
| 5 | Immobilized cultures | 87 |
| 4 | Cell line preservation and characterization | 95 |
| 2 | Cell line banking | 96 |
| 3 | Cell freezing, and quantitation of recovery | 97 |
| 4 | Cell line characterizations | 104 |
| 5 | Computerized cell source and databases | 145 |
| 5 | Separation of viable cells by centrifugal elutriation | 149 |
| 2 | Cell separation methods | 149 |
| 3 | The centrifugal elutriator | 150 |
| 4 | Technical protocols | 155 |
| 5 | Collection and interpretation of data | 158 |
| 6 | Future developments | 160 |
| 6 | Flow cytometry | 165 |
| 2 | Principles of operation | 166 |
| 3 | Data output and analysis | 181 |
| 4 | Quality control | 186 |
| 5 | Cell sorting | 190 |
| 6 | Preparation of samples | 197 |
| 7 | Examples of types of assay | 200 |
| 7 | Organ culture | 213 |
| 2 | Survey of organ culture techniques | 213 |
| 3 | Watchglass technique | 215 |
| 4 | The Maximow single slide technique | 221 |
| 5 | Agar gel techniques | 223 |
| 6 | Embryo culture for teratological studies | 231 |
| 7 | Grid technique | 233 |
| 8 | Organ culture of tissues: examples | 241 |
| 9 | Long-term cultivation of human adult tissues | 246 |
| 10 | Preparation of organ cultures for light microscopy | 249 |
| 11 | Autoradiography | 253 |
| 12 | Guide to quantitation of results | 257 |
| 13 | Advantages and limitations of organ culture | 258 |
| 8 | Cytotoxicity and viability assays | 263 |
| 2 | Background | 264 |
| 3 | Specific techniques | 265 |
| 4 | Endpoints | 272 |
| 5 | Assay comparisons | 280 |
| 6 | Technical protocols | 281 |
| 7 | Interpretation of results | 295 |
| 8 | Pitfalls and trouble shooting | 298 |
| 9 | Automation and future developments | 300 |
| 9 | In situ hybridization | 305 |
| 2 | Hybridization technique | 305 |
| 3 | Applications of in situ hybridization | 309 |
| 4 | The choice of probe | 309 |
| 5 | The choice of label | 310 |
| 6 | Sensitivity | 310 |
| 7 | Negative controls | 310 |
| 8 | Detailed protocols for in situ hybridization | 311 |
| A1 General reagents | 317 | |
| A2 Suppliers of specialist items | 319 | |
| Index | 325 |
Overview
This book has sold over 10,000 copies since its publication in 1986, and remains one of the most popular titles in the Practical Approach Series. This new, thoroughly updated edition details the recent progress made in this field. There have been significant advances in areas such as serum-free media, scale-up, and flow cytometry. As these techniques have developed as tools for cell biologists, their availability to the non-specialist has also increased dramatically. Use of the tetrazolium salt MTT as a ...