Methods in Cell-Matrix Adhesion
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Table of contents
- Cover
- Copyright Pageiv
- Contentsv
- Contributorsxi
- Acknowledgmentsxv
- Part I: Preface and Perspectives on Cell–Matrix Adhesion1
- Preface3
- Cell–Matrix Interactions: The View from the Outside7
- Matrix and Meaning13
- Cell–Matrix Adhesion Research and the Development of Biotherapeutics17
- Part II: Matrix Methodologies25
- Chapter 1. Detection and Purification of Instructive Extracellular Matrix Components with Monoclonal27
- I. Introduction28
- II. Methods for Detecting ECM Components That Instruct Cell Responses in Reporter Cells32
- III. Preparation of Mabs for Identification of Instructive ECM41
- IV. Purification of Instructive ECM with Mabs and Interactions with Reporter Cells47
- V. Summary49
- References49
- Chapter 2. Isolation and Purification of Proteoglycans53
- I. Overview and Scope53
- II. Extraction of Proteoglycans55
- III. Radiolabeling of Proteoglycans for Ease of Purification57
- IV. Anion-Exchange Chromatography57
- V. Hydrophobic Interaction Chromatography59
- VI. Gel Filtration Chromatography60
- VII. Immunoaffinity Chromatography61
- VIII. Quantification and Characterization63
- References66
- Chapter 3. Expression of Recombinant Matrix Components Using Baculoviruses69
- I. Introduction: The Importance of Recombinant Extracellular Matrix Molecules for Protein Studies70
- II. Requirements and Rationale for Development of a General Protein Expression System70
- III. Expression in Insect Cells Using pCOCO74
- IV. Results Obtained from the COCO System78
- References80
- Chapter 4. Heparan Sulfate–Growth Factor Interactions83
- I. Introduction: HS Regulation of Growth Factor Signaling83
- II. Methods for Identifying the Role of HS in Growth Factor Binding and Signaling89
- III. Identification of Regulatory HS95
- IV. Analyzing the Structure of HS: General Considerations and Methodologies102
- References104
- Chapter 5. Analysis of Basement Membrane Self-Assembly and Cellular Interactions with Native and Rec111
- I. Introduction111
- II. Native and Recombinant Basement Membrane Proteins114
- III. Analysis of Self-Assembly in Vitro124
- IV. Analysis of Cell-Surface Contributions to Basement Membrane Assembly135
- V. Approaches to Evaluating ECM Molecules for Assembly Interactions136
- VI. Integration of Genetic and Molecular Approaches137
- References138
- Chapter 6. Preparation and Analysis of Synthetic Multicomponent Extracellular Matrix145
- I. Introduction to the ECM145
- II. Development of Synthetic Matrices147
- III. Discussion155
- References158
- Chapter 7. Analysis of Matrix Dynamics by Atomic Force Microscopy163
- I. Introduction164
- II. General Experimental Design165
- III. Probe Microscopy in Fluid: AFM Imaging168
- IV. Probe Microscopy in Fluid: Molecular Pulling/Force Spectroscopy174
- V. Probe Microscopy in Air: AFM Imaging182
- VI. Conclusion188
- References188
- Chapter 8. Analysis of Matrix Degradation195
- I. Introduction195
- II. Experimental Approaches and Protocols196
- References204
- Part III: Adhesion Receptor Methodologies207
- Chapter 9. Expression Cloning Strategies for the Identification of Adhesion Molecules209
- I. Introduction209
- II. Importance of Methodology212
- III. General Considerations212
- IV. Protocols214
- V. Points of Variation218
- VI. Critical Controls219
- VII. Potential Use in Disease/Drug Screening Applications219
- References220
- Chapter 10. Purification of Integrins and Characterization of Integrin-Associated Proteins223
- I. Introduction224
- II. Isolation of Integrins: General Considerations and Points of Variation225
- III. Characterization of Integrin-Associated Proteins through mAb Production: General Considerations234
- References241
- Chapter 11. Methods for Analysis of the Integrin Ligand Binding Event247
- I. Introduction247
- II. Application of Affinity Cross-Linking to Study of the Integrin Ligand Binding Pocket248
- III. Microtiter-Based Integrin Binding Assays250
- IV. Measuring Ligand Binding to Integrins with Surface Plasmon Resonance254
- V. Analyzing Integrin Redox Status256
- References258
- Chapter 12. Intracellular Coupling of Adhesion Receptors: Molecular Proximity Measurements261
- I. Introduction and Applications261
- II. Fluorescent Lifetime Imaging Microscopy (FLIM)265
- References276
- Part IV: Functional Applications of Cell–Matrix Adhesion in Molecular Cell Biology279
- Chapter 13. Functional Analysis of Cell Adhesion: Quantitation of Cell–Matrix Attachment281
- I. Introduction281
- II. General Concepts in Cell Adhesion Assays283
- III. Cell Attachment Assays286
- IV. Cell Spreading Assays288
- V. Direct Binding Assays289
- VI. Cell Migration Assays290
- VII. Conclusions293
- References293
- Chapter 14. Measurements of Glycosaminoglycan-Based Cell Interactions297
- I. Introduction297
- II. Cell Adhesion Assay299
- III. Cell Invasion Assay302
- References307
- Chapter 15. Applications of Adhesion Molecule Gene Knockout Cell Lines309
- I. Introduction310
- II. Derivation of Somatic Cell Lines311
- III. ES Cell Lines Carrying Homozygous Mutations of Integrin Adhesion Molecule Genes315
- IV. Studies with Adhesion Receptor Null Cell Lines317
- V. Studies with Extracellular Matrix Component Null Cell Lines321
- VI. Summary322
- References322
- Chapter 16. Flexible Polyacrylamide Substrata for the Analysis of Mechanical Interactions at Cell–325
- I. Introduction326
- II. Preparation of the Polyacrylamide Substratum327
- III. Characterization of the Gel330
- IV. Data Collection for the Analysis of Traction Forces332
- V. Analysis of Traction Forces334
- VI. Other Applications Using Polyacrylamide Substrata336
- VII. Summary338
- References338
- Chapter 17. Cell Migration in Slice Cultures341
- I. Introduction341
- II. Somitic Migration342
- III. Migration from the Subventricular Zone to the Olfactory Bulb349
- IV. Axonal Outgrowth353
- V. Conclusions and Perspectives356
- References357
- Chapter 18. Application of Cell Adhesion to Study Signaling Networks359
- I. Introduction359
- II. Applications361
- III. Methods362
- References382
- Chapter 19. Use of Micropatterned Adhesive Surfaces for Control of Cell Behavior385
- I. Introduction385
- II. Materials and Instrumentation392
- III. Procedures394
- IV. Comments399
- V. Pitfalls399
- References400
- Chapter 20. Adenoviral-Mediated Gene Transfer in Two-Dimensional and Three-Dimensional Cultures of M403
- I. Introduction404
- II. Using Collagen I as a Three-Dimensional Matrix for Cell Culture405
- III. Using Reconstituted Basement Membrane as a Three-Dimensional Matrix for Cell Culture407
- IV. Preparing Mouse Mammary Epithelial Cell Cultures409
- V. Preparing Recombinant Adenovirus for Infecting Mammary Epithelial Cells409
- VI. Infecting Mammary Epithelial Cell Cultures with Adenovirus419
- References421
- Part V: General Information425
- Appendix A: List of Suppliers427
- Appendix B: Relevant Microarray Dataset Experiments431
- Appendix C: Web Site Resources of Interest435
- Index441
- Volumes in Series455
Critically acclaimed for more than 25 years, the Methods in Cell Biology series provides an indispensable tool for the researcher. Each volume is carefully edited by experts to contain state-of-the-art reviews and step-by-step protocols. Techniques are described completely so that methods are made accessible to users. This volume, Methods of Cell-Matrix Adhesion, contains integrated coverage on cell-matrix adhesion methods. It brings the classical methodologies and the latest techniques together in one concise volume. This coverage includes experimental protocols and their conceptual background for all aspects of cell-matrix adhesion research: the extracellular matrix, adhesion receptors, and the growing number of functional applications of matrix-adhesion in molecular cell biology. Also covered is the purification of the extracellular matrix to functional analyses of cellular responses.