Culture of animal cells: a manual of basic technique
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Buch |
| Sprache: | English |
| Veröffentlicht: |
New York [u.a.]
Wiley-Liss
2000
|
| Ausgabe: | 4. ed. |
| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | XXVI, 577 S. Ill., graph. Darst. |
| ISBN: | 0471348899 |
Internformat
MARC
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| 245 | 1 | 0 | |a Culture of animal cells |b a manual of basic technique |c R. Ian Freshney |
| 250 | |a 4. ed. | ||
| 264 | 1 | |a New York [u.a.] |b Wiley-Liss |c 2000 | |
| 300 | |a XXVI, 577 S. |b Ill., graph. Darst. | ||
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Datensatz im Suchindex
| _version_ | 1804127901962993664 |
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| adam_text | Figures, xvii
Color Plates, xxi
Preface, xxiii
Abbreviations, xxv
11ntroduction, 1
Background, 1
Advantages of Tissue Culture, 4
Control of the Environment, 4
Characterization and Homogeneity of Sample, 4
Economy, Scale, and Mechanization, 4
In Vivo Modeling, 5
Limitations, 5
Expertise, 5
Quantity, 5
Dedifferentiation and Selection, 5
Origin of Cells, 5
Instability, 6
Major Differences In Vitro, 6
Types of Tissue Culture, 6
2 Biology of Cultured Cells, 9
The Culture Environment, 9
Cell Adhesion, 9
Cell Adhesion Molecules, 10
Cytoskeleton, 10
Cell Proliferation, 11
Cell Cycle, 11
Control of Cell Proliferation, 11
Differentiation, 11
Maintenance of Differentiation, 12
Dedifferentiation, 12
Energy Metabolism, 13
Initiation of the Culture, 1 5
Evolution of Cell Lines, 15
Senescence, 16
The Development of Continuous Cell Lines, 16
Origin of Cultured Cells, 16
3 Design and Layout, 19
Planning, 19
Construction and Services, 20
Layout, 22
Sterile Handling Area, 23
Incubation, 24
Hot Room, 24
Service Bench, 27
Preparation, 27
Wash Up, 27
Storage, 28
4 Equipment, 31
Requirements of a Tissue Culture Laboratory
Essential Equipment, 31
Laminar Flow Hood, 31
Incubator, 32
vii
viii 4fr contents
CO2 Incubator, 34
Sterilizer, 34
Refrigerators and Freezers, 35
Inverted Microscope, 37
Washing Up, 37
Sterilizing and Drying, 38
Water Purification, 38
Centrifuge, 39
Cryostorage Container, 39
Balance, 39
Hemocytometer, 40
Beneficial Equipment, 40
Cell Counter, 40
Aspiration Pump, 40
pH Meter, 41
Conductivity Meter, 41
Osmometer, 41
Upright Microscope, 41
Dissecting Microscope, 41
Temperature Recording, 41
Magnetic Stirrer, 41
Roller Racks, 41
Fluid Handling, 42
Useful Additional Equipment, 46
Low Temperature Freezer, 46
Glassware Washing Machine, 46
Video Camera and Monitor, 47
Colony Counter, 48
Controlled Rate Freezer, 48
Centrifugal Elutriator, 48
Flow Cytometer, 48
Consumable Items, 48
Pipettes, 48
Culture Vessels, 49
Sterile Containers, 49
Syringes and Needles, 49
Sterilization Filters, 49
5 Aseptic Technique, 51
Objectives of Aseptic Technique, 51
Maintaining Sterility, 51
Elements of Aseptic Environment, 52
Quiet Area, 52
Work Surface, 52
Personal Hygiene, 53
Reagents and Media, 53
Cultures, 54
Sterile Handling, 55
Swabbing, 55
Capping, 55
Flaming, 55
Handling Bottles and Flasks, 55
Pipetting, 56
Pouring, 56
Laminar Flow, 56
Standard Procedure, 58
Protocol 5.1. Working in Laminar Flow, 58
Protocol 5.2. Working on the Open Bench, 60
Petri Dishes and Multiwell Plates, 61
Protocol 5.3. Handling Dishes or Plates, 62
Apparatus and Equipment, 62
Incubators, 62
Boxed Cultures, 62
Gassing with CO2, 62
Training, 62
6 Safety, 65
Risk Assessment, 65
Standard Operating Procedures, 65
Safety Regulations, 68
General Safety, 68
Operator, 68
Equipment, 68
Glassware and Sharp Items, 68
Chemical Toxicity, 70
Gases, 71
Liquid Nitrogen, 71
Fire, 72
Radiation, 72
Ingestion, 72
Labeled Reagents, 72
High Energy Sources, 72
Biohazards, 73
Levels of Containment, 73
Human Biopsy Material, 73
Genetic Manipulation, 75
Disposal, 76
7 Culture Vessels, 77
The Substrate, 77
Attachment and Growth, 77
Substrate Materials, 77
Choice of Culture Vessel, 78
Cell Yield, 78
Suspension Culture, 80
Venting, 80
Sampling and Analysis, 81
Uneven Growth, 82
Cost, 82
Specialized Systems, 83
Permeable Supports, 83
Treated Surfaces, 83
Matrix Coating, 83
Protocol 7.1. Preparation of ECM, 84
Feeder Layers, 85
Three dimensional Matrices, 85
Alternative Artificial Substrates, 85
Nonadhesive Substrates, 86
Liquid Gel or Liquid Liquid Interfaces, 86
8 Media, IIS
Development of Media, 89
Physicochemical Properties, 89
pH, 89
Protocol 8.1. Preparations of pH Standards, 90
CO2 and Bicarbonate, 90
Buffering, 91
Oxygen, 91
Osmolality, 92
Temperature, 92
Viscosity, 93
Surface Tension and Foaming, 93
Balanced Salt Solutions, 93
Complete Media, 94
Amino Acids, 94
Vitamins, 95
Salts, 95
Glucose, 95
Organic Supplements, 95
Hormones and Growth Factors, 95
Antibiotics, 95
Serum, 99
Protein, 99
Growth Factors, 99
Hormones, 100
Nutrients and Metabolites, 101
Lipids, 101
Minerals, 101
Inhibitors, 101
Selection of Medium and Serum, 101
Batch Reservation, 103
Testing Serum, 103
Heat Inactivation, 103
Other Supplements, 103
Embryo Extract, 104
Conditioned Medium, 104
9 Serum Free Media, 104
Disadvantages of Serum, 10S
Advantages of Serum Free Media, 110
Selective Media, 110
Regulation of Proliferation and
Differentiation, 111
Disadvantages of Serum Free Media, 111
Replacement of Serum, 111
CONTENTS ? JX
Subculture, 111
Hormones, 111
Growth Factors, 116
Nutrients in Serum, 116
Proteins and Polyamines, 116
Matrix, 116
Selection of Serum Free Medium, 116
Commercially Available Serum Free Media, 117
Serum Substitutes, 117
Development of Serum Free Medium, 117
Preparation of Serum Free Medium, 120
Conclusions, 120
10 Preparation and Sterilization, 121
Apparatus, 121
Glassware, 121
Protocol 10.1. Preparation and Sterilization of
Glassware, 122
Pipettes, 125
Protocol 10.2. Preparation and Sterilization of
Pipettes, 125
Screw Caps, 127
Protocol 10.3. Preparation and Sterilization of
Screw Caps, 128
Selection of Detergent, 128
Miscellaneous Equipment, 129
Reusable Sterilizing Filters, 130
Protocol 10.4. Sterilizing Filter Assemblies, 130
Reagents and Media, 130
Water, 131
Balanced Salt Solutions, 132
Protocol 10.5. Preparation of BSS, 132
Media, 133
Protocol 10.6. Preparation of Medium from IX
Stock, 133
Protocol 10.7. Preparation of Medium from 10X
Concentrate, 1 34
Powdered Media, 136
Protocol 10.8. Preparation of Medium from
Powder, 136
Customized Medium, 137
Protocol 10.9. Preparation of Customized
Medium, 137
Sterilization, 139
Sterile Filtration, 139
Protocol 10.10. Sterile Filtration with Syringe tip
Filter, 140
Protocol 10.11. Sterile Filtration with Filter
Flask, 141
Protocol 10.12. Sterile Filtration with Small In line
Filter, 142
Protocol 10.13. Sterile Filtration with Large In line
Filter, 142
X ^^ CONTENTS
Serum, 143
Protocol 10.14. Collection and Sterilization of
Serum, 143
Protocol 10.15. Serum Dialysis, 144
Preparation and Sterilization of Other
Reagents, 146
Control, Testing, and Storage of Media, 146
Quality Control, 146
Sterility Testing, 146
Culture Testing, 146
Storage, 147
11 Primary Culture, 149
Types of Primary Cell Culture, 149
Isolation of the Tissue, 149
Mouse Embryo Cell Culture, 150
Protocol 11.1. Isolation of Mouse Embryos, 150
Chick Embryo Cell Culture, 152
Protocol 11.2. Isolation of Chick Embryos, 152
Human Biopsy Material, 153
Protocol 11.3. Human Biopsies, 157
Primary Culture, 157
Primary Explant, 157
Protocol 11.4. Primary Explants, 158
Enzymatic Disaggregation, 159
Protocol 11.5. Warm Trypsin, 161
Trypsinization with Cold Preexposure, 163
Protocol 11.6. Cold Trypsin, 163
Chick Embryo Organ Rudiments, 165
Protocol 11.7. Chick Embryo Organ
Rudiments, 165
Other Enzymatic Procedures, 169
Collagenase, 169
Protocol 11.8. Disaggregation in Collagenase, 169
Mechanical Disaggregation, 171
Protocol 11.9. Mechanical Disaggregation by
Sieving, 173
Separation of Viable and Nonviable Cells, 173
Protocol 11.10. Enrichment of Viable Cells, 173
Primary Records, 175
12 Cell Lines, 177
Nomenclature, 177
Subculture and Propagation, 177
Immortalization of Cell Lines, 180
Cell Line Designations, 180
Selection of Cell Line, 181
Routine Maintenance, 181
Cell Morphology, 181
Replacement of Medium, 181
Volume, Depth, and Surface Area, 181
Protocol 12.1. Changing the Medium or
Feeding, 182
Holding Medium, 183
Subculture, 183
Criteria for Subculture, 183
Protocol 12.2. Subculture, 184
Split Ratios and Growth Cycle, 188
Cell Concentration at Subculture, 188
Propagation in Suspension, 189
Subculture of Suspension Culture, 189
Protocol 12.3. Subculture in Suspension,
189
Use of Antibiotics, 191
Maintenance Records, 193
13 Cloning and Selection, 195
Cloning, 195
Protocol 13.1. Dilution Cloning, 196
Stimulation of Plating Efficiency, 198
Improving Clonal Growth, 198
Conditioned Medium, 199
Protocol 13.2. Preparation of Conditioned
Medium, 199
Feeder Layers, 199
Protocol 13.3. Preparation of Feeder Layers,
200
Suspension Cloning, 200
Protocol 13.4. Cloning in Agar, 200
Protocol 13.5. Cloning in Methocel, 202
Isolation of Clones, 204
Protocol 13.6. Isolation of Clones with Cloning
Rings, 206
Protocol 13.7. Isolating Cell Colonies by
Irradiation, 206
Other Isolation Techniques for Monolayer
Clones, 206
Suspension Clones, 206
Protocol 13.8. Isolation of Suspension
Clones, 206
Replica Plating, 207
Selective Inhibitors, 207
Isolation of Genetic Variants, 208
Protocol 13.9. Methotrexate Resistance and DHFR
Amplification, 208
Interaction with Susbstrate, 211
Selective Adhesion, 211
Selective Detachment, 211
Nature of Substrate, 211
Selective Feeder Layers, 212
Semisolid Media, 213
14 Cell Separation, 215
Cell Density and Isopyknic Sedimentation, 215
Protocol 14.1. Cell Separation by Density
Gradient, 215
Variations, 218
Antibody Based Techniques, 218
Immune Panning, 218
Magnetic Sorting, 219
Protocol 14.2. Magnetic Activated Cell Sorting
(MACS), 219
Cell Size and Sedimentation Velocity, 222
Centrifugal Elutriation, 222
Fluorescence Activated Cell Sorting, 223
Other Techniques, 225
Beginner s Approach to Cell Separation, 226
15 Characterization, 229
The Need for Characterization, 229
Species Identification, 230
Lineage or Tissue Markers, 230
Unique Markers, 231
Transformation, 231
Morphology, 231
Microscopy, 234
Protocol 15.1. Using an Inverted Microscope, 234
Staining, 234
Protocol 15.2. Staining with Giemsa, 235
Protocol 15.3. Staining with Crystal Violet, 236
Culture Vessels for Cytology: Monolayer
Cultures, 236
Preparation of Suspension Cultures for
Cytology, 236
Protocol 15.4. Smear Preparation, 236
Protocol 15.5. Cytocentrifuge, 236
Protocol 15.6. Filtration Cytology, 238
Photography, 239
Protocol 15.7. Microscope Photography on
Film, 239
Electronic Image Recording, 240
Protocol 15.8. Electronic Images from
Microscope, 241
Chromosome Analysis, 241
Protocol 15.9. Chromosome Preparations, 242
Chromosome Banding, 242
Chromosome Analysis, 243
Chromosome Painting, 245
DNA Content, 245
DNA Hybridization, 246
DNA Fingerprinting, 246
Protocol 15.10. Multilocus DNA Fingerprinting of
Cell Lines, 247
RNA and Protein, 249
CONTENTS +¦ XJ
Enzyme Activity, 251
Isoenzymes, 251
Protocol 15.11. Isoenzyme Analysis, 252
Antigenic Markers, 254
Protocol 15.12. Indirect Immunofluorescence, 256
Differentiation, 257
Authentication, 257
16 Differentiation, 259
Expression of In Vivo Phenotype, 259
Dedifferentiation, 259
Stages of Commitment and Differentiation, 259
Proliferation and Differentiation, 260
Commitment and Lineage, 260
Markers of Differentiation, 261
Induction of Differentiation, 262
Soluble Inducers, 262
Cell Interaction, 262
Paracrine Growth Factors, 264
Negatively Acting Paracrine Factors, 265
Cell Matrix Interactions, 265
Polarity and Cell Shape, 266
Differentiation and Malignancy, 266
Practical Aspects, 266
17 Transformation, 269
Role in Cell Line Characterization, 269
What is Transformation?, 269
Genetic Instability, 269
Chromosomal Aberrations, 271
DNA Content, 271
Immortalization, 271
Control of Senescence, 272
Immortalization with Viral Genes, 273
Immortalization of Human Fibroblasts, 274
Protocol 17.1. Fibroblast Immortalization, 274
Telomerase induced Immortalization, 277
Transgenic Mouse, 277
Aberrant Growth Control, 277
Anchorage Independence, 277
Contact Inhibition, 278
Protocol 17.2. Density Limitation of Cell
Proliferation, 279
Serum Dependence, 279
Tumorigenicity, 280
Malignancy, 280
Tumorigenesis, 281
Invasiveness, 281
Angiogenesis, 282
Plasminogen Activator, 283
XJi •#? CONTENTS
18 Contamination, 285
Sources of Contamination, 285
Operator Technique, 285
Environment, 285
Use and Maintenance of Laminar flow Hood
Humid Incubators, 288
Protocol 18.1. Cleaning Incubators, 288
Cold Stores, 289
Sterile Materials, 289
Imported Cell Lines and Biopsies, 289
Quarantine, 289
Types of Microbial Contamination, 289
Monitoring for Contamination, 289
Visible Microbial Contamination, 289
Mycoplasma, 290
Protocol 18.2. Fluorescence Detection of
Mycoplasma, 292
Viral Contamination, 294
Eradication of Contamination, 294
Bacteria, Fungi, and Yeasts, 294
Protocol 18.3. Eradication of Microbial
Contamination, 294
Eradication of Mycoplasma, 294
Eradication of Viral Contamination, 295
Persistent Contamination, 295
Cross Contamination, 295
Conclusions, 296
19 Cryopreservation, 297
Need for Cryopreservation, 297
Rationale for Freezing, 297
Preservation, 297
Selection of Cell Line, 297
Standardization of Culture Conditions, 297
Stages of Cryopreservation, 298
Protocol 19.1. Freezing Cells, 299
Cooling Rate, 302
Cryofreezers, 302
Freezer Records, 303
Protocol 19.2. Thawing Frozen Cells, 303
Serial Replacement, 307
Cell Banks, 307
Transporting Cells, 308
Frozen Ampules, 308
Living Cultures, 308
20 Quantitation, 309
Cell Counting, 309
Hemocytometer, 309
Protocol 20.1. Cell Counting by
Hemocytometer, 309
Electronic Counting, 312
Protocol 20.2. Electronic Cell Counting, 312
Cell Sizing, 313
Stained Monolayers, 313
Cell Weight, 314
DNA Content, 314
Protocol 20.3. DNA Estimation by Hoechst
33258, 314
Protein, 314
Solubilization of Sample, 315
Protocol 20.4. Protein Estimation by Bradford
Method, 315
Rates of Synthesis, 315
DNA Synthesis, 315
Protocol 20.5. DNA Synthesis, 315
Protein Synthesis, 316
Protocol 20.6 Protein Synthesis, 316
Preparation of Samples for Enzyme Assay and
Immunoassay, 317
Cytometry, 317
In situ Labeling, 317
Flow Cytometry, 318
Replicate Sampling, 318
Data Acquisition, 318
Data Analysis, 318
Cell Proliferation, 319
Experimental Design, 319
Growth Cycle, 319
Protocol 20.7. Growth Curve, Monolayer, 319
Suspension Cultures, 321
Protocol 20.8. Growth Curve, Suspension, 321
Analysis of the Growth Cycle, 322
Plating Efficiency, 323
Protocol 20.9. Plating Efficiency, 324
Automatic Colony Counting, 325
Labeling Index, 325
Protocol 20.10. Labeling Index with [3H]
thymidine, 325
Growth Fraction, 327
Protocol 20.11. Determination of Growth
Fraction, 327
Mitotic Index, 327
Division Index, 327
Cell Cycle Time (Generation Time), 327
Cell Migration, 328
21 Cytotoxicity, 329
Introduction, 329
In Vitro Limitations, 330
Nature of the Assay, 330
Viability, 330
Protocol 21.1. Estimation of Viability by Dye
Exclusion, 331
Protocol 21.2. Estimation of Viability by Dye
Uptake, 331
Survival, 332
Protocol 21.3. Clonogenic Assay, 332
Cell Proliferation Assays, 335
Metabolic Assays, 335
Microtitration Assays, 335
Protocol 21.4. MTT Based Cytotoxicity Assay, 336
Comparison of Microtitration with Cloning, 338
Drug Interaction, 340
Anticancer Drug Screening, 340
Predictive Testing, 340
Transformation, 340
Protocol 21.5. Sister Chromatic) Exchange, 341
Carcinogenicity, 343
Inflammation, 343
22 Specialized Cells. 345
Epithelial Cells, 345
Epidermis, 346
Protocol 22.1. Epidermal Keratinocytes, 347
Cornea, 349
Protocol 22.2. Corneal Epithelial Cells, 350
Breast, 351
Protocol 22.3. Mammary Epithelium, 351
Cervix, 352
Protocol 22.4. Cervical Epithelium, 352
Gastrointestinal Tract, 355
Protocol 22.5. Colorectal Epithelium, 355
Liver, 357
Protocol 22.6. Isolation of Rat Hepatocytes, 357
Pancreas, 358
Protocol 22.7. Pancreatic Epithelium, 358
Kidney, 359
Protocol 22.8. Kidney Epithelium, 360
Bronchial and Tracheal Epithelium, 361
Protocol 22.9. Bronchial and Tracheal
Epithelium, 361
Prostate, 362
Protocol 22.10. Prostatic Epithelium, 362
Mensenchymal Cells, 364
Connective Tissue, 364
Adipose Tissue, 364
Protocol 22.11. Primary Culture of Adipose
Cells, 365
Muscle, 366
Protocol 22.12. Skeletal Muscle, 366
Cartilage, 367
Protocol 22.13. Chondrocytes in Alginate
Beads, 368
Bone, 370
Protocol 22.14. Osteoblasts, 370
Endothelium, 372
CONTENTS ^ XJii
Protocol 22.15. Vascular Endothelial Cells,
372
Neuroectodermal Cells, 373
Neurons, 373
Protocol 22.16. Cerebellar Granule Cells, 373
Glial Cells, 374
Protocol 22.17. Olfactory Bulb Ensheathing
Cells, 376
Endocrine Cells, 378
Melanocytes, 378
Protocol 22.18. Melanocytes, 378
Hematopoietic Cells, 380
Long Term Bone Marrow Cultures, 381
Protocol 22.19. Long term Hematopoietic
Cell Cultures from Bone Marrow,
381
Hematopoietic Colony Forming Assays, 382
Protocol 22.20. Hematopoietic Colony forming
Assays, 382
Conads, 384
Germ Cells, 384
23 Tumor Cells, 385
Sampling, 386
Disaggregation, 387
Primary Culture, 387
Characterization, 387
Development of Cell Lines, 388
Continuous Cell Lines, 388
Selective Culture, 388
Selective Media, 389
Confluent Feeder Layers, 389
Protocol 23.1. Growth on Confluent Feeder
Layers, 390
Suspension Cloning, 391
Histotypic Culture, 391
Spheroids, 391
Xenografts, 391
Preservation of Tissue by Freezing, 391
Protocol 23.2. Freezing Biopsies, 391
Specific Tumor Types, 392
Breast, 392
Lung, 392
Colon, 392
Pancreas, 392
Ovary, 392
Prostate, 393
Skin, 393
Cervix, 393
Neuroblastoma, 393
Seminoma, 393
XJV ¦+• CONTENTS
24 Drganotypic Culture, 395
Cell Interaction and Phenotypic Expression, 395
Reciprocal Interactions, 395 Choice of
Models, 396
Organ Culture, 396
Gas and Nutrient Exchange, 396
Structural Integrity, 396
Growth and Differentiation, 397
Limitations of Organ Culture, 397
Types of Organ Culture, 397
Protocol 24.1. Organ Culture, 397
Histotypic Culture, 399
Gel and Sponge Techniques, 399
Hollow Fibers, 399
Spheroids, 399
Protocol 24.2. Spheroids, 400
Immobilization of Living Cells in Alginate, 402
Protocol 24.3. Alginate Encapsulation, 402
Filter Well Inserts, 403
Protocol 24.4. Filter Well Inserts, 404
Cultures of Neuronal Aggregates, 405
Protocol 24.5. Neuronal
Aggregates, 405z Organotypic Culture, 406
25 Scale Up, 4D7
Scale up Suspension, 407
Protocol 25.1. Stirred 4 liter Batch Suspension
Culture, 407
Continuous Culture, 409
Scale and Complexity, 410
Mixing and Aeration, 410
Scale up Monolayer, 000
Multisurface Propagators, 412
Protocol 25.2. Nunclon Cell Factory, 413
Multiarray Disks, Spirals, and Tubes, 414
Roller Culture, 414
Protocol 25.3. Roller Bottle Culture, 414
Microcarriers, 416
Protocol 25.4. Microcarriers, 417
Perfused Monolayer Culture, 419
Monitoring Growth, 420
26 Specialized Techniques, 423
Lymphocyte Preparation, 423
Protocol 26.1. Preparation of Lymphocytes, 423
Blast Transformation, 424
Protocol 26.2. PHA Stimulation of
Lymphocytes, 424
Autoradiography, 424
Protocol 26.3. Microautoradiography, 425
Time Lapse Recording, 429
Protocol 26.4. Time Lapse Video, 430
Confocal Microscopy, 431
Cell Synchrony, 431
Cell Separation, 431
Blockade, 431
Culture of Amniocytes, 432
Protocol 26.5. Culture of Amniocytes, 432
Culture of Cells from Poikilotherms, 437
Fish Cells, 437
Reagents and Media for Protocols 26.6 26.8, 438
Protocol 26.6. Fibroblast Feeder Layers from
Zebrafish Embryos, 439
Protocol 26.7. Primary Cultures from Zebrafish
Embryos, 439
Protocol 26.8. Cell Lines from Zebrafish
Embryos, 440
Insect Cells, 440
Protocol 26.9. Propagation of Insect Cells, 440
11 Molecular Techniques, 443
Molecular Biology in Cell Culture, 443
In Situ Molecular Hybridization, 443
Analysis of RNA Gene Expression by in Situ
Hybridization, 443
Protocol 27.1. Autoradiographic in Situ
Hybridization, 443
Fluorescence in Situ Hybridization in the Analysis
of Genes and Chromosomes, 447
Protocol 27.2. FISH Using Single Copy Genomic
Probes and Chromosome Painting, 447
Somatic Cell Fusion, 449
Protocol 27.3. Cell Hybridization, 449
Selection of Hybrid Clones, 451
Nuclear Transfer, 451
Monochromosomal Transfer, 451
Production of Monoclonal Antibodies, 452
Protocol 27.4. Production of Monoclonal
Antibodies, 452
DNA Transfer, 455
Coprecipitation with Calcium Phosphate, 456
Protocol 27.5. Stable DNA Transfection with
Calcium Phosphate, 456
Lipofection, 457
Protocol 27.6. Transient Transfection by
Lipofection, 457
Electroporation, 458
Protocol 27.7. Stable Transfection by
Electroporation, 459
Other DNA Transfer Methods, 460
Reporter Genes, 460
Protocol 27.8. In Situ Staining for /3
galactosidase, 461
Protocol 27.9. Chloramphenicol Acetyltransferase
(CAT) Assay, 461
28 Problem Solving, 463
Slow Cell Growth, 463
Is the problem restricted to your own stocks or are
other people having similar problems?, 463
If the problem is more general and other
researchers are having difficulty as well, check
shared facilities and reagents, 464
Have any changes occurred in the laboratory?, 464
Medium, 464
Choice of Medium, 465
Selection of the correct medium is critical, 465
Unstable Reagents, 466
Glutamine, 466
Serum, 466
Other Constituents, 466
Trypsin, 466
Purity of Constituents, 466
Plastics, 466
Glassware, 466
Wash Up, 466
Contamination, 467
Single User, 467
Widespread, 467
Identification of Contamination, 468
Chemical Contamination, 469
Primary Culture, 469
Poor Take in Primary Culture, 469
Wrong Cells, 470
Contamination, 470
Cloning, 470
Poor Plating Efficiency, 470
Diffuse Colonies, 471
Too Many Colonies per Dish, 471
Nonrandom Distribution, 471
CONTENTS ¦+¦ XV
Nonadherent Cells, 471
Differentiation, 471 Cells Do Not
Differentiate, 471
Loss of Product Formation, 471
Feeding, 471
Clones, 471
Subculture, 471
Cell Cycle Phase at Subculture, 472
Senescence, 472
Medium, 472
Uneven Growth, 472
Cross Contamination, 472
Symptoms, 472
Prophylaxis, 472
Cure, 472
Cryopreservation, 472
Poor Recovery, 472
Changed Appearance After Cryopreservation, 473
Contamination, 473
Loss of Stock, 473
Cell Counting, 473
Hemocytometer, 473
Electronic, 473
Viability, 474
Morphological Appearance, 474
Testing Viability, 474
Cytotoxicity, 474
In Conclusion, 475
Reagent Appendix, 477
Trade Index, 483
Sources of Materials, 483
Suppliers and Other Resources, 488
Glossary, 517
References, 523
General Textbooks, 561
Useful Journals, 563
Index, 565
|
| any_adam_object | 1 |
| author | Freshney, Robert Ian 1938-2019 |
| author_GND | (DE-588)1055981578 |
| author_facet | Freshney, Robert Ian 1938-2019 |
| author_role | aut |
| author_sort | Freshney, Robert Ian 1938-2019 |
| author_variant | r i f ri rif |
| building | Verbundindex |
| bvnumber | BV013201404 |
| callnumber-first | Q - Science |
| callnumber-label | QH585 |
| callnumber-raw | QH585.2 |
| callnumber-search | QH585.2 |
| callnumber-sort | QH 3585.2 |
| callnumber-subject | QH - Natural History and Biology |
| classification_rvk | WX 6600 WX 6603 |
| classification_tum | BIO 768f BIO 770f CIT 972f BIO 655f |
| ctrlnum | (OCoLC)246210294 (DE-599)BVBBV013201404 |
| dewey-full | 571.638 |
| dewey-hundreds | 500 - Natural sciences and mathematics |
| dewey-ones | 571 - Physiology & related subjects |
| dewey-raw | 571.638 |
| dewey-search | 571.638 |
| dewey-sort | 3571.638 |
| dewey-tens | 570 - Biology |
| discipline | Biologie Chemie-Ingenieurwesen Biotechnologie |
| edition | 4. ed. |
| format | Book |
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| id | DE-604.BV013201404 |
| illustrated | Illustrated |
| indexdate | 2024-07-09T18:40:43Z |
| institution | BVB |
| isbn | 0471348899 |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-008994453 |
| oclc_num | 246210294 |
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| physical | XXVI, 577 S. Ill., graph. Darst. |
| publishDate | 2000 |
| publishDateSearch | 2000 |
| publishDateSort | 2000 |
| publisher | Wiley-Liss |
| record_format | marc |
| spelling | Freshney, Robert Ian 1938-2019 Verfasser (DE-588)1055981578 aut Culture of animal cells a manual of basic technique R. Ian Freshney 4. ed. New York [u.a.] Wiley-Liss 2000 XXVI, 577 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Gewebekultur (DE-588)4157245-2 gnd rswk-swf Tierzelle (DE-588)4185509-7 gnd rswk-swf Methode (DE-588)4038971-6 gnd rswk-swf Tiere (DE-588)4060087-7 gnd rswk-swf Zellkultur (DE-588)4067547-6 gnd rswk-swf Gewebekultur (DE-588)4157245-2 s Tierzelle (DE-588)4185509-7 s DE-604 Zellkultur (DE-588)4067547-6 s Tiere (DE-588)4060087-7 s Methode (DE-588)4038971-6 s 1\p DE-604 2\p DE-604 HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=008994453&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis 1\p cgwrk 20201028 DE-101 https://d-nb.info/provenance/plan#cgwrk 2\p cgwrk 20201028 DE-101 https://d-nb.info/provenance/plan#cgwrk |
| spellingShingle | Freshney, Robert Ian 1938-2019 Culture of animal cells a manual of basic technique Gewebekultur (DE-588)4157245-2 gnd Tierzelle (DE-588)4185509-7 gnd Methode (DE-588)4038971-6 gnd Tiere (DE-588)4060087-7 gnd Zellkultur (DE-588)4067547-6 gnd |
| subject_GND | (DE-588)4157245-2 (DE-588)4185509-7 (DE-588)4038971-6 (DE-588)4060087-7 (DE-588)4067547-6 |
| title | Culture of animal cells a manual of basic technique |
| title_auth | Culture of animal cells a manual of basic technique |
| title_exact_search | Culture of animal cells a manual of basic technique |
| title_full | Culture of animal cells a manual of basic technique R. Ian Freshney |
| title_fullStr | Culture of animal cells a manual of basic technique R. Ian Freshney |
| title_full_unstemmed | Culture of animal cells a manual of basic technique R. Ian Freshney |
| title_short | Culture of animal cells |
| title_sort | culture of animal cells a manual of basic technique |
| title_sub | a manual of basic technique |
| topic | Gewebekultur (DE-588)4157245-2 gnd Tierzelle (DE-588)4185509-7 gnd Methode (DE-588)4038971-6 gnd Tiere (DE-588)4060087-7 gnd Zellkultur (DE-588)4067547-6 gnd |
| topic_facet | Gewebekultur Tierzelle Methode Tiere Zellkultur |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=008994453&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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