Culture of animal cells: a manual of basic technique and specialized applications
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| Format: | Buch |
| Sprache: | English |
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Wiley-Blackwell
2010
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| Ausgabe: | 6. ed. |
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| Beschreibung: | Zusatz zum Sachtitel lautete bis zur 5. Aufl. nur "a manual of basic technique" |
| Beschreibung: | XXXI, 732 S. Ill., graph. Darst. |
| ISBN: | 9780470528129 |
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Datensatz im Suchindex
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| adam_text | IMAGE 1
A MANUAL OF BASIC TECHNIQUE
AND SPECIALIZED APPLICATIONS SIXTH EDITION
R. IAN FRESHNEY CANCER RESEARCH UK CENTRE FOR ONCOLOGY AND APPLIED
PHARMACOLOGY DIVISION OF CANCER SCIENCES AND MOLECULAR PHARMACOLOGY
UNIVERSITY OF GLASGOW
)WILEY-BLACKWELL A JOHN WILEY & SONS, INC., PUBLICATION
IMAGE 2
LIST OF FIGURES, XIX
LIST OF COLOR PLATES, XXIII LIST OF PROTOCOLS, XXV PREFACE AND
ACKNOWLEDGEMENTS, XXVII ABBREVIATIONS, XXIX
1.
1.1. 1.2.
1.3.
1.4. 1.5.
HISTORICAL BACKGROUND, 1 ADVANTAGES OF TISSUE CULTURE, 6 1.2.1.
1.2.2.
1.2.3. 1.2.4.
CONTROL OF THE ENVIRONMENT, 6 CHARACTERIZATION AND HOMOGENEITY OF
SAMPLES, 6 ECONOMY, SCALE, AND MECHANIZATION, 6
IN VITRO MODELING OF IN VIVO CONDITIONS, 7 LIMITATIONS, 7 1.3.1.
1.3.2. 1.3.3. 1.3.4.
1.3.5. MAJOR TVDES I
EXPERTISE, 7 QUANTITY, 7 DEDIFFERENTIATION AND SELECTION, 8 ORIGIN OF
CELLS, 8
INSTABILITY, 8 DIFFERENCES IN VITRO, 8 OF TISSUE CULTURE. 8
2.3.
2.4.
2.5. 2.6. 2.7.
2.2.2 2.2.3 2.2.4 2.2.5
CELL 2.3.1 2.3.2
INTERCELLULAR JUNCTIONS, 12 EXTRACELLULAR MATRIX, 13 CYTOSKELETON, 14
CELL MOTILITY, 14 PROLIFERATION, 15 CELL CYCLE, 15
CONTROL OF CELL PROLIFERATION, 15 DIFFERENTIATION, 16 2.4.1 2.4.2 CELL
MAINTENANCE OF DIFFERENTIATION, 17 DEDIFFERENTIATION, 17 SIGNALING, 17
ENERGY METABOLISM, 19 ORIGIN OF CULTURED CELLS, 20 2.7.1 2.7.2
2.7.3 2.7.4
INITIATION OF THE CULTURE, 21 EVOLUTION OF CELL LINES, 21 SENESCENCE, 22
TRANSFORMATION AND THE DEVELOPMENT
OF CONTINUOUS CELL LINES, 22
3. LABORATORY DESIGN, LAYOUT,
AND EQUIPMENT 25
I BIOLOGY OF CULTURED CELLS, 2.1. THE CULTURE ENVIRONMENT, 11 2.2. CELL
ADHESION, 11 2.2.1. CELL ADHESION MOLECULES, 11
3.1. LAYOUT, FURNISHING, AND SERVICES, 25 3.1.1. REQUIREMENTS, 25 3.1.2.
SERVICES, 28 3.1.3. VENTILATION, 30 3.2. LAYOUT, 30
3.2.1. STERILE HANDLING AREA, 30 3.2.2. LAMINAR FLOW, 30 3.2.3. SERVICE
BENCH, 30
3.2.4. QUARANTINE AND CONTAINMENT, 30
VII
IMAGE 3
VIII
O CONTENTS
3.2.5. INCUBATION, 31
3.2.6. PREPARATION AREA, 33 3.2.7. STORAGE, 34
4. EQUIPMENT AND MATERIALS, 37 4.1. REQUIREMENTS OF A TISSUE CULTURE
LABORATORY, 37 4.2. ASEPTIC AREA, 37
4.2.1. LAMINAR-FLOW HOOD, 37 4.2.2. SERVICE CARTS, 41 4.2.3. STERILE
LIQUID HANDLING-PIPETTING AND DISPENSING, 41 4.2.4. INVERTED MICROSCOPE,
45 4.2.5. CCD CAMERA AND MONITOR, 46 4.2.6.. DISSECTING MICROSCOPE, 46
4.2.7. CENTRIFUGE, 47 4.2.8. CELL COUNTING, 47 4.3. INCUBATION AND
CULTURE, 47
4.3.1.., INCUBATOR, 47 4.3.2. HUMID CO 2 INCUBATOR, 48 4.3.3.
TEMPERATURE RECORDER, 48 4.3.4. ROLLER RACKS, 49
4.3.5. MAGNETIC STIRRER, 50 4.3.6. CULTURE VESSELS, 50 4.4. PREPARATION
AND STERILIZATION, 50 4.4.1. WASHUP, 50
4.4.2. PREPARATION OF MEDIA AND REAGENTS, 51 4.4.3. STERILIZATION, 52
4.5. STORAGE, 53 4.5.1. CONSUMABLES, 53
4.5.2. REFRIGERATORS AND FREEZERS, 54 4.5.3. CRYOSTORAGE CONTAINERS, 55
4.5.4. CONTROLLED-RATE FREEZER, 55 4.6. SUPPLEMENTARY LABORATORY
EQUIPMENT, 55
4.6.1. COMPUTERS AND NETWORKS, 55 4.6.2. UPRIGHT MICROSCOPE, 55 4.6.3.
LOW-TEMPERATURE FREEZER, 56 4.6.4. CONFOCAL MICROSCOPE, 56 4.6.5. PCR
THERMAL CYCLER, 56 4.7. SPECIALIZED EQUIPMENT, 56
4.7.1. MICROINJECTION FACILITIES, 56 4.7.2. COLONY COUNTER, 56 4.7.3.
CENTRIFUGAL ELUTRIATOR, 56 4.7.4. FLOW CYTOMETER, 56
5.2.2. QUIET AREA, 60 5.2.3. WORK SURFACE, 61 5.2.4. PERSONAL HYGIENE,
61 5.2.5. REAGENTS AND MEDIA, 61
5.2.6. CULTURES, 61 5.3. STERILE HANDLING, 61 5.3.1. SWABBING, 61
5.3.2. CAPPING, 63 5.3.3. FLAMING, 63 5.3.4. HANDLING BOTTLES AND
FLASKS, 64 5.3.5. PIPETTING, 64
5.3.6. POURING, 65 5.4. STANDARD PROCEDURE, 65 PROTOCOL 5.1. ASEPTIC
TECHNIQUE IN VERTICAL LAMINAR FLOW, 65
PROTOCOL 5.2. WORKING ON THE OPEN BENCH, 67 PROTOCOL 5.3. HANDLING
DISHES OR PLATES, 69 5.5. APPARATUS AND EQUIPMENT, 69 5.5.1. INCUBATORS,
69
5.5.2. BOXED CULTURES, 70 5.5.3. GASSING WITH CCB, 70
6. SAFETY, BIOETHICS, AND VALIDATION, 71 6.1. LABORATORY SAFETY, 71 6.2.
RISK ASSESSMENT, 71 6.3. STANDARD OPERATING PROCEDURES, 73 6.4. SAFETY
REGULATIONS, 73 6.5. GENERAL SAFETY, 74
6.5.1. OPERATOR, 74
5. ASEPTIC TECHNIQUE, 57 5.1. OBJECTIVES OF ASEPTIC TECHNIQUE, 57 5.1.1.
RISK OF CONTAMINATION, 57 5.1.2. MAINTAINING STERILITY, 57 5.2. ELEMENTS
OF ASEPTIC ENVIRONMENT, 58
5.2.1. LAMINAR FLOW, 58
6.6. 6.7.
6.8.
6.5.2. 6.5.3. 6.5.4. 6.5.5. 6.5.6.
6.5.7. FIRE, 78 IONIZING 6.7.1. 6.7.2.
6.7.3. 6.7.4.
EQUIPMENT, 74 GLASSWARE AND SHARP ITEMS, 74 CHEMICAL TOXICITY, 76 GASES,
76 LIQUID NITROGEN, 76 BURNS, 78
RADIATION, 78 INGESTION, 78 DISPOSAL OF RADIOACTIVE WASTE, 78
IRRADIATION FROM LABELED REAGENTS, 78
IRRADIATION FROM HIGH-ENERGY SOURCES, 79 BIOHAZARDS, 79 6.8.1. 6.8.2.
6.8.3. 6.8.4. 6.8.5. 6.8.6.
LEVELS OF BIOLOGICAL CONTAINMENT, 79 MICROBIOLOGICAL SAFETY CABINETS
(MSCS), 79 HUMAN BIOPSY MATERIAL, 79
GENETIC MANIPULATION, 84 DISPOSAL OF BIOHAZARDOUS WASTE, 85 FUMIGATION,
85 6.9. BIOETHICS, 86
6.9.1. ANIMAL TISSUE, 86 6.9.2. HUMAN TISSUE, 86
IMAGE 4
CONTENTS
JX
6.10. QUALITY ASSURANCE, 87 6.10.1. PROCEDURES, 87 6.10.2. QUALITY
CONTROL (QC), 87 6.11. VALIDATION, 87
6.11.1. AUTHENTICATION, 87 6.11.2. PROVENANCE, 88 6.11.3. CONTAMINATION,
88
7. CULTURE VESSELS AND SUBSTRATES, 89 7.1. THE SUBSTRATE, 89 7.1.1.
ATTACHMENT AND GROWTH, 89 7.1.2. COMMON SUBSTRATE MATERIALS, 89
7.1.3. ALTERNATIVE SUBSTRATES, 90 7.2. TREATED SURFACES, 90 7.2.1.
/SUBSTRATE COATING, 90 PROTOCOL 7.1. PREPARATION OF ECM, 91
7.2.2S FEEDER LAYERS, 91 7.2.3. NONADHESIVE SUBSTRATES, 91 7.3. CHOICE
OF CULTURE VESSEL, 91 7.3.1. CELL YIELD, 93
7.3.2. SUSPENSION CULTURE, 93 7.3.3. VENTING, 94 7.3.4. SAMPLING AND
ANALYSIS, 94 7.3.5. UNEVEN GROWTH, 95
7.3.6. COST, 96 7.4. SPECIALIZED SYSTEMS, 96 7.4.1. PERMEABLE SUPPORTS,
96 7.4.2. THREE-DIMENSIONAL MATRICES, 97
8. DEFINED MEDIA AND SUPPLEMENTS, 99 8.1. 8.2. DEVELOPMENT OF MEDIA, 99
PHYSICOCHEMICAL PROPERTIES, 99 8.2.1. PROTOCOL 8. 1.
8.3.
8.4.
8.2.2. 8.2.3. 8.2.4. 8.2.5.
8.2.6. 8.2.7. 8.2.8.
PH, 99 PREPARATION OFPH STANDARDS, 100 CO2 AND BICARBONATE, 100
BUFFERING, 101 OXYGEN, 105 OSMOLALITY, 106 TEMPERATURE, 106 VISCOSITY,
107
SURFACE TENSION AND FOAMING, 107 BALANCED SALT SOLUTIONS, 107 COMPLETE
MEDIA, 107 8.4.1.
8.4.2. 8.4.3. 8.4.4. 8.4.5. 8.4.6. 8.4.7.
AMINO ACIDS, 108 VITAMINS, 108 SALTS, 108 GLUCOSE, 108
ORGANIC SUPPLEMENTS, 108 HORMONES AND GROWTH FACTORS, 109 ANTIBIOTICS,
109
8.5. SERUM, 109 8.5.1. PROTEIN, 109 8.5.2. GROWTH FACTORS, 111 8.5.3.
HORMONES, 111
8.5.4. NUTRIENTS AND METABOLITES, 111 8.5.5. LIPIDS, 111 8.5.6.
MINERALS, 111 8.5.7. INHIBITORS, 111 8.6. SELECTION OF MEDIUM AND SERUM,
111
8.6.1. BATCH RESERVATION, 112 8.6.2. TESTING SERUM, 113 8.6.3. HEAT
INACTIVATION, 114 8.7. OTHER SUPPLEMENTS, 114 8.7.1. AMINO ACID
HYDROLYSATES, 114 8.7.2. EMBRYO EXTRACT, 114
8.7.3. CONDITIONED MEDIUM, 114
9. SERUM-FREE MEDIA, 115 9.1. DISADVANTAGES OF SERUM, 115 9.2.
ADVANTAGES OF SERUM-FREE MEDIA, 121 9.2.1. DEFINITION OF STANDARD
MEDIUM, 121
9.2.2. SELECTIVE MEDIA, 121 9.2.3. REGULATION OF PROLIFERATION AND
DIFFERENTIATION, 121 9.3. DISADVANTAGES OF SERUM-FREE MEDIA, 122 9.4.
REPLACEMENT OF SERUM, 122
9.4.1. COMMERCIALLY AVAILABLE SERUM-FREE MEDIA, 122 9.4.2. SERUM
SUBSTITUTES, 122 9.4.3. SERUM-FREE SUBCULTURE, 123
9.4.4. HORMONES, 123 9.4.5. GROWTH FACTORS, 123 9.4.6. NUTRIENTS IN
SERUM, 124 9.4.7. PROTEINS AND POLYAMINES, 124 9.4.8. VISCOSITY, 124
9.5. SELECTION OF SERUM-FREE MEDIUM, 124
9.5.1. CELL OR PRODUCT SPECIFICITY, 124 9.5.2. ADAPTATION TO SERUM-FREE
MEDIA, 124 9.6. DEVELOPMENT OF SERUM-FREE MEDIUM, 124 9.7. PREPARATION
OF SERUM-FREE MEDIUM, 129 9.8. ANIMAL PROTEIN-FREE MEDIA, 129 9.9.
CONCLUSIONS, 132
10. PREPARATION AND STERILIZATION, 133 10.1. PREPARATION OF REAGENTS AND
MATERIALS, 133 10.2. STERILIZATION OF APPARATUS AND LIQUIDS, 133 10.3.
APPARATUS, 134
10.3.1. GLASSWARE, 134 PROTOCOL 10.1. PREPARATION AND STERILIZATION OF
GLASSWARE, 135
IMAGE 5
O
CONTENTS
10.3.2. GLASS PIPETTES, 136 PROTOCOL 10.2. PREPARATION AND STERILIZATION
OF GLASS PIPETTES, 136
10.3.3. SCREW CAPS, 137 PROTOCOL 10.3. PREPARATION AND STERILIZATION OF
SCREW CAPS, 137
10.3.4. SELECTION OF DETERGENT, 138 10.3.5. MISCELLANEOUS EQUIPMENT, 139
10.3.6. REUSABLE STERILIZING FILTERS, 139 PROTOCOL 10.4. STERILIZING
FILTER ASSEMBLIES, 139 10.4. REAGENTS AND MEDIA, 140
10.4.1. WATER, 140 PROTOCOL 10.5. PREPARATION AND STERILIZATION OF
ULTRAPURE WATER (UPW), 142 10.4.2. MAINTENANCE OF WATER PURIFIER, 143
10.4.3. BALANCED SALT SOLUTIONS, 143 PROTOCOL 10.6. .PREPARATION AND
STERILIZATION OF D-PBSA, 144
10.4.4. PREPARATION AND STERILIZATION OF MEDIA, 144 PROTOCOL 10,7.
PREPARATION OF MEDIUM FROM LX STOCK, 145 PROTOCOL 10.8. PREPARATION OF
MEDIUM FROM LOX
CONCENTRATE, 146 10.4.5. POWDERED MEDIA, 148 PROTOCOL 10.9. PREPARATION
OF MEDIUM FROM POWDER, 149
10.4.6. CUSTOMIZED MEDIUM, 150 PROTOCOL 10.10. PREPARATION OF CUSTOMIZED
MEDIUM, 150 10.5. STERILIZATION OF MEDIA, 151
10.5.1. AUTOCLAVABLE MEDIA, 151 10.5.2. STERILE FILTRATION, 151 PROTOCOL
10.11. STERILE FILTRATION WITH SYRINGE-TIP FILTER, 153 PROTOCOL 10.12.
STERILE FILTRATION WITH VACUUM FILTER
FLASK, 155 PROTOCOL 10.13. STERILE FILTRATION WITH SMALL IN-LINE FILTER,
156 PROTOCOL 10.14. STERILE FILTRATION WITH LARGE IN-LINE
FILTER, 156 10.5.3. SERUM, 157 PROTOCOL 10.15. COLLECTION AND
STERILIZATION OF SERUM, 157 PROTOCOL 10.16. DIALYSIS OF SERUM, 160
10.5.4. PREPARATION AND STERILIZATION OF OTHER REAGENTS, 160 10.6.
CONTROL, TESTING, AND STORAGE OF MEDIA, 160 10.6.1. QUALITY CONTROL, 160
10.6.2. STERILITY TESTING, 161 10.6.3. CULTURE TESTING, 161 10.6.4.
STORAGE, 162
1 1. PRIMARY CULTURE, 163 11.1. INITIATION OF A PRIMARY CELL CULTURE,
163 11.1.1. ENZYMES USED IN DISAGGREGATION, 163 11.1.2. COMMON FEATURES
OF
DISAGGREGATION, 164
11.2. ISOLATION OF THE TISSUE, 164 11.2.1. MOUSE EMBRYO, 164 PROTOCOL
11.1. ISOLATION OF MOUSE EMBRYOS, 164 11.2.2. CHICK EMBRYO, 166 PROTOCOL
11.2. ISOLATION OF CHICK EMBRYOS, 166
11.2.3. HUMAN BIOPSY MATERIAL, 168 PROTOCOL 11.3. HANDLING HUMAN
BIOPSIES, 170 11.3. TYPES OF PRIMARY CULTURE, 170 11.3.1. PRIMARY
EXPLANTATION, 170
PROTOCOL 11.4. PRIMARY EXPLANTS, 170 11.3.2. ENZYMATIC DISAGGREGATION,
173 11.3.3. WARMTRYPSIN, 173 PROTOCOL 11.5. TISSUE DISAGGREGATION IN
WARM
TRYPSIN, 173 11.3.4. TRYPSINIZATION WITH COLD PREEXPOSURE, 175 PROTOCOL
11.6. TISSUE DISAGGREGATION IN COLD
TRYPSIN, 176 11.3.5. CHICK EMBRYO ORGAN RUDIMENTS, 177 PROTOCOL 11.7.
CHICK EMBRYO ORGAN RUDIMENTS, 177 11.3.6. OTHER ENZYMATIC PROCEDURES,
181
11.3.7. COLLAGENASE, 181 PROTOCOL 11.8. TISSUE DISAGGREGATION IN
COLLAGENASE, 181 11.3.8. MECHANICAL DISAGGREGATION, 183 PROTOCOL 11.9.
MECHANICAL DISAGGREGATION
BY SIEVING, 183 11.3.9. SEPARATION OF VIABLE AND NONVIABLE CELLS, 184
PROTOCOL 11.10. ENRICHMENT OF VIABLE CELLS, 184
11.3.10. PRIMARY CULTURE IN SUMMARY, 186 11.3.11. PRIMARY RECORDS, 186
12. SUBCULTURE AND CELL LINES, 187 12.1. SUBCULTURE AND PROPAGATION, 187
12.1.1. CROSS-CONTAMINATION AND MISIDENTIFICATION, 187
12.1.2. MYCOPLASMA CONTAMINATION, 191 12.1.3. TERMINOLOGY, 191 12.1.4.
NAMING A CEU LINE, 192
12.1.5. CULTURE AGE, 192 12.2. CHOOSING A CELL LINE, 193 12.3. ROUTINE
MAINTENANCE, 193
12.3.1. SIGNIFICANCE OF CELL MORPHOLOGY, 193
IMAGE 6
CONTENTS
XI
12.3.2. REPLACEMENT OF MEDIUM, 194 12.3.3. STANDARD FEEDING PROTOCOL,
195 PROTOCOL 12.1. FEEDING A MONOLAYER CULTURE IN FLASKS, 195 PROTOCOL
12.2. FEEDING A MONOLAYER CULTURE IN PLATES OR DISHES, 196 12.4.
SUBCULTURE, 196
12.4.1. CNTERIA FOR SUBCULTURE, 197 12.4.2. TYPICAL SUBCULTURE PROTOCOL
FOR CELLS GROWN AS A MONOLAYER, 199 PROTOCOL 12.3. SUBCULTURE OF
MONOLAYER CELLS, 199 12.4.3. GROWTH CYCLE AND SPLIT RATIOS, 201 12.4.4.
CELL CONCENTRATION AT SUBCULTURE, 202 12.4.5. PROPAGATION IN SUSPENSION,
202 12.4.6. SUBCULTURE OF CELLS GROWING
IN SUSPENSION, 202
PROTOCOL 12.4. SUBCULTURE OF SUSPENSION CELLS, 203 12.4.7.
STANDARDIZATION OF CULTURE CONDITIONS, 204 12.4.8. USE OF ANTIBIOTICS,
205 12.4.9. MAINTENANCE RECORDS, 206
13.8.1. SELECTIVE ADHESION, 224 13.8.2. SELECTIVE DETACHMENT, 224
13.8.3. NATURE OF SUBSTRATE, 225 13.8.4. SELECTIVE FEEDER LAYERS, 225
13.8.5. SELECTION BY SEMISOLID MEDIA, 225
14. CELL SEPARATION, 227
13. CLONING AND SELECTION, 207 13.1. CELL CLONING, 207 PROTOCOL 13.1.
DILUTION CLONING, 208 13.2. STIMULATION OF PLATING EFFICIENCY, 209
13.2.1. CONDITIONS THAT IMPROVE CLONAL GROWTH, 211 13.2.2. CONDITIONED
MEDIUM, 212 PROTOCOL 13.2. PREPARATION OF CONDITIONED MEDIUM, 212
13.2.3. FEEDER LAYERS, 213 PROTOCOL 13.3. PREPARATION OF FEEDER LAYERS,
213 13.3. SUSPENSION CLONING, 2 14 PROTOCOL 13.4. CLONING IN AGAR, 214
PROTOCOL 13.5. CLONING IN METHOCEL, 217 13.4. ISOLATION OF CLONES, 218
PROTOCOL 13.6. ISOLATION OF CLONES WITH CLONING RINGS, 218
PROTOCOL 13.7. ISOLATING CELL COLONIES BY IRRADIATION, 219 13.4.1. OTHER
ISOLATION TECHNIQUES FOR MONOLAYER CLONES, 220
13.4.2. SUSPENSION CLONES, 221 PROTOCOL 13.8. ISOLATION OF SUSPENSION
CLONES, 221 13.5. REPLICA PLATING, 221 13.6. SELECTIVE INHIBITORS, 221
13.7. ISOLATION OF GENETIC VARIANTS, 223 PROTOCOL 13.9. METHOTREXATE
RESISTANCE AND DHFR AMPLIFICATION, 223
13.8. INTERACTION WITH SUBSTRATE, 224
14.1. CELL DENSITY AND ISOPYKNIC SEDIMENTATION, 227 PROTOCOL 14.1. CELL
SEPARATION BY CENTRIFUGATION ON A DENSITY GRADIENT, 227 14.2. CELL SIZE
AND SEDIMENTATION VELOCITY, 230
14.2.1. UNIT GRAVITY SEDIMENTATION, 230 14.2.2. CENTRIFUGAL ELUTRIATION,
230 14.3. ANTIBODY-BASED TECHNIQUES, 232 14.3.1. IMMUNE PANNING, 232
14.3.2. MAGNETIC SORTING, 233 PROTOCOL 14.2. MAGNET-ACTIVATED CELL
SORTING (MACS), 234 14.4. FLUORESCENCE-ACTIVATED CELL SORTING, 234 14.5.
OTHER TECHNIQUES, 236
14.6. BEGINNER S APPROACH TO CELL SEPARATION, 237
15. CHARACTERIZATION, 239 15.1. THE NEED FOR CHARACTERIZATION, 239 15.2.
AUTHENTICATION, 239 15.3. RECORD KEEPING AND PROVENANCE, 240 15.4.
PARAMETERS OF CHARACTERIZATION, 240
15.4.1. SPECIES IDENTIFICATION, 240 15.4.2. LINEAGE OR TISSUE MARKERS,
241 15.4.3. UNIQUE MARKERS, 242 15.4.4. TRANSFORMATION, 242 15.5. CELL
MORPHOLOGY, 242
15.5.1. MICROSCOPY, 247 PROTOCOL 15.1. USING AN INVERTED MICROSCOPE, 248
15.5.2. STAINING, 248 PROTOCOL 15.2. STAINING WITH GIEMSA, 249 PROTOCOL
15.3. STAINING WITH CRYSTAL VIOLET, 249
15.5.3. CULTURE VESSELS FOR CYTOLOGY: MONOLAYER CULTURES, 250 15.5.4.
PREPARATION OF SUSPENSION CULTURE FOR CYTOLOGY, 250 PROTOCOL 15.4.
PREPARATION OF SUSPENSION CELLS
FOR CYTOLOGY BY CYTOCENTRIFUGE, 251 PROTOCOL 15.5. FILTRATION CYTOLOGY,
251 15.5.5. PHOTOMICROGRAPHY, 252
IMAGE 7
XII
O CONTENTS
PROTOCOL 15.6. DIGITAL PHOTOGRAPHY ON A MICROSCOPE, 252
15.6. CONFOCAL MICROSCOPY, 253 15.7. CHROMOSOME CONTENT, 253 PROTOCOL
15.7. CHROMOSOME PREPARATIONS, 253 15.7.1. CHROMOSOME BANDING, 255
15.7.2. CHROMOSOME ANALYSIS, 256 15.8. DNA ANALYSIS, 256 15.8.1. DNA
HYBRIDIZATION, 256
15.8.2. DNA FINGERPRINTING, 257 15.8.3. T NA PROFILING, 258 PROTOCOL
15.8. DNA STR PROFILING OF CELL LINES, 259 15.9. RNA AND PROTEIN
EXPRESSION, 261 15.10. ENZYME ACTIVITY, 261
15.10.1. ISOENZYMES, 262 15.10.2. ISOENZYME ELECTROPHORESIS WITH
AUTHENTIKIT, 263 PROTOCOL 15.9. _ ISOENZYME ANALYSIS, 263 15.11.
ANTIGENFC MARKERS, 267
15.11.1. IMMUNOSTAINING, 267 PROTOCOL 1^.10. INDIRECT
IMMUNOFIUORESCENCE, 267 15.11..2. IMMUNOANALYSIS, 268 15.12.
DIFFERENTIATION, 268
17.4.2. IMMORTALIZATION WITH VIRAL GENES, 283 17.4.3. IMMORTALIZATION OF
HUMAN FIBROBLASTS, 283 PROTOCOL 17.1. FIBROBLAST IMMORTALIZATION, 284
17.4.4. TELOMERASE-INDUCED IMMORTALIZATION, 287 PROTOCOL 17.2.
IMMORTALIZATION OF HUMAN STEM AND PRIMARY CELLS BY TELOMERASE, 287
17.4.5. LYMPHOCYTE IMMORTALIZATION, 290 17.4.6. TRANSGENIC MOUSE, 290
17.5. ABERRANT GROWTH CONTROL, 290 17.5.1. ANCHORAGE INDEPENDENCE, 290
17.5.2. CONTACT INHIBITION, 291 PROTOCOL 17.3. DENSITY LIMITATION OF
CELL PROLIFERATION, 291
17.5.3. SERUM DEPENDENCE, 292 17.5.4. ONCOGENES, 293 17.6.
TUMORIGENICITY, 293 17.6.1. MALIGNANCY, 293
17.6.2. TUMOR TRANSPLANTATION, 293 17.6.3. INVASIVENESS, 294 17.6.4.
ANGIOGENESIS, 294 PROTOCOL 1 7. 4. IN VITRO ANGIOGENESIS ASSAY, 295
17.6.5. PLASMINOGEN ACTIVATOR, 297
16. DIFFERENTIATION, 269 16.1. EXPRESSION OF THE IN VIVO PHENOTYPE, 269
16.1.1. DEDIFFERENTIATION, 269 16.1.2. LINEAGE SELECTION, 269 16.2.
STAGES OF DIFFERENTIATION, 270 16.3. PROLIFERATION AND DIFFERENTIATION,
270
16.4. COMMITMENT AND LINEAGE, 270 16.5. STEM CELL PLASTICITY, 271 16.6.
MARKERS OF DIFFERENTIATION, 272 16.7. INDUCTION OF DIFFERENTIATION, 272
16.7.1. CELL INTERACTION, 273 16.7.2. SYSTEMIC FACTORS, 274 16.7.3.
CELL-MATRIX INTERACTIONS, 277 16.7.4. POLARITY AND CELL SHAPE, 277
16.7.5. OXYGEN TENSION, 277 16.8. DIFFERENTIATION AND MALIGNANCY, 278
16.9. PRACTICAL ASPECTS, 278
17. TRANSFORMATION AND IMMORTALIZATION, 279 17.1. ROLE IN CELL LINE
CHARACTERIZATION, 279 17.2. WHAT IS TRANSFORMATION?, 279 17.3. GENETIC
INSTABILITY AND HETEROGENEITY, 279
17.3.1. GENETIC INSTABILITY, 279 17.3.2. CHROMOSOMAL ABERRATIONS, 281
17.4. IMMORTALIZATION, 281 17.4.1. CONTROL OF SENESCENCE, 282
IB. CONTAMINATION, 299 18.1. SOURCES OF CONTAMINATION, 299 18.1.1.
OPERATOR TECHNIQUE, 299 18.1.2. ENVIRONMENT, 299
18.1.3. USE AND MAINTENANCE OF LAMINAR-FLOW HOOD, 299 18.1.4. HUMID
INCUBATORS, 300 PROTOCOL 18.1. CLEANING INCUBATORS, 300
18.1.5. COLD STORES, 301 18.1.6. STERILE MATERIALS, 301 18.1.7. IMPORTED
CELL LINES AND BIOPSIES, 301 18.1.8. QUARANTINE, 301 18.2. TYPES OF
MICROBIAL CONTAMINATION, 301 18.3. MONITORING FOR CONTAMINATION, 301
18.3.1. VISIBLE MICROBIAL CONTAMINATION, 304 18.3.2. MYCOPLASMA, 305
18.3.3. FLUORESCENCE STAINING FOR MYCOPLASMA, 306
PROTOCOL 18.2. FLUORESCENCE DETECTION OF MYCOPLASMA, 306 18.3.4. PCR FOR
MYCOPLASMA, 307 PROTOCOL 18.3. DETECTION OF MYCOPLASMA BY PCR, 307
18.3.5. ALTERNATIVE METHODS FOR DETECTING MYCOPLASMA, 310 18.3.6.
MYCOPLASMA DETECTION SERVICES, 311 18.3.7. VIRAL CONTAMINATION, 311
IMAGE 8
CONTENTS 2 XIII
18.4. DISPOSAL OF CONTAMINATED CULTURES, 311 18.5. ERADICATION OF
CONTAMINATION, 311 18.5.1. BACTERIA, FUNGI, AND YEASTS, 311 PROTOCOL
18.4. ERADICATION OF MICROBIAL
CONTAMINATION, 311 18.5.2. ERADICATION OF MYCOPLASMA, 312 PROTOCOL 18.5.
ERADICATION OF MYCOPLASMA CONTAMINATION, 312
18.5.3. ERADICATION OF VIRAL CONTAMINATION, 313 18.5.4. PERSISTENT
CONTAMINATION, 313 18.6. CROSS-CONTAMINATION, 315 18.7. CONCLUSIONS, 315
20. QUANTITATION, 335
19. CRYOPRESERVATION, 317 19.1. RATIONALE FOR FREEZING, 317 19.2.
CONSIDERATIONS BEFORE CRYOPRESERVATION, 317
19.2.1. VALIDATION, 317 19.2.2. WHEN TO FREEZE, 318 19.3. PRINCIPLES OF
CRYOPRESERVATION, 318 19.3.1. THEORETICAL BACKGROUND TO CELL
FREEZING, 318
19.3.2. CELL CONCENTRATION, 318 19.3.3. FREEZING MEDIUM, 318 19.3.4.
COOLING RATE, 319 19.3.5. AMPOULES, 320
19.3.6. CRYOFREEZERS, 321 19.3.7. FREEZING CULTURED CELLS, 324 PROTOCOL
19.1. FREEZING CELLS, 324 19.3.8. FREEZER RECORDS, 325
19.3.9. THAWING STORED AMPOULES, 325 PROTOCOL 19.2. THAWING FROZEN
CELLS, 326 19.3.10. FREEZING FLASKS, 327 19.4. VITRIFICATION, 327
19.4.1. CRYOPRESERVATION OF HES CELLS, 328 PROTOCOL 19.3.
CRYOPRESERVATION OF HES CELLS BY VITRIFICATION, 328 19.4.2. THAWING HES
CELLS, 330
PROTOCOL 19.4. THAWING HES CELLS CRYOPRESERVED BY VITRIFICATION, 330
19.5. DESIGN AND CONTROL OF FREEZER STOCKS, 331 19.5.1. FREEZER
INVENTORY CONTROL, 331
19.5.2. SERIAL REPLACEMENT OF CULTURE STOCK, 332 19.6. CELL BANKS, 332
19.7. TRANSPORTING CELLS, 333
19.7.1. FROZEN AMPOULES, 333 19.7.2. LIVING CULTURES, 333
20.1. CELL COUNTING, 335 20.1.1. HEMOCYTOMETER, 335 PROTOCOL 20.1. CELL
COUNTING BY HEMOCYTOMETER, 335 20.1.2. ELECTRONIC COUNTING, 339
PROTOCOL 20.2. ELECTRONIC CELL COUNTING BY ELECTRICAL RESISTANCE, 340
20.1.3. STAINED MONOLAYERS, 342 20.1.4. FLOW CYTOMETRY, 343 20.2. CELL
WEIGHT, 344 20.3. DNA CONTENT, 344
PROTOCOL 20.3. DNA ESTIMATION BY HOECHST 33258, 345 20.4. PROTEIN, 345
20.4.1. SOLUBILIZATION OF SAMPLE, 345
20.4.2. BRADFORD ASSAY, 345 PROTOCOL 20.4. PROTEIN ESTIMATION BY THE
BRADFORD METHOD, 345 20.5. RATES OF SYNTHESIS, 346
20.5.1. DNA SYNTHESIS, 346 PROTOCOL 20.5. ESTIMATION OF DNA SYNTHESIS BY
[ 3H]THYMIDINE INCORPORATION, 346
20.5.2. PROTEIN SYNTHESIS, 347 PROTOCOL 20.6. PROTEIN SYNTHESIS, 347
20.6. PREPARATION OF SAMPLES FOR ENZYME ASSAY AND IMMUNOASSAY, 348 20.7.
CYTOMETRY, 348
20.7.1. IN SITU LABELING, 348 20.7.2. FLOW CYTOMETRY, 348 20.8.
REPLICATE SAMPLING, 348 20.8.1. DATA ACQUISITION, 349
20.8.2. DATA ANALYSIS, 349 20.9. CELL PROLIFERATION, 349 20.9.1.
EXPERIMENTAL DESIGN, 349 20.9.2. GROWTH CYCLE, 350
PROTOCOL 20.7. GROWTH CURVE WITH A MONOLAYER IN FLASKS, 351 PROTOCOL
20.8. GROWTH CURVE WITH A MONOLAYER IN MULTIWELL PLATES, 352
20.9.3. ANALYSIS OF MONOLAYER GROWTH CURVES, 353 20.9.4. MEDIUM VOLUME,
CELL CONCENTRATION, AND CELL DENSITY, 353 20.9.5. SUSPENSION CULTURES,
355 PROTOCOL 20.9. GROWTH CURVE WITH CELLS IN
SUSPENSION, 355 20.9.6. PHASES OF THE GROWTH CYCLE, 355 20.9.7.
DERIVATIVES FROM THE GROWTH CURVE, 357 20.10. PLATING EFFICIENCY, 357
PROTOCOL 20.10. DETERMINATION OF PLATING EFFICIENCY, 358 20.10.1.
ANALYSIS OF COLONY FORMATION, 359
IMAGE 9
XIV
O CONTENTS
20.10.2. AUTOMATIC COLONY COUNTING, 359 20.11. LABELING INDEX, 360
PROTOCOL 20.11. LABELING INDEX WITH [ 3 HJTHYMIDINE, 361
20.11.1. GROWTH FRACTION, 361 PROTOCOL 20.12. DETERMINATION OF GROWTH
FRACTION, 362 20.11.2. MITOTIC INDEX, 363 20.11.3. DIVISION INDEX, 363
20.12. CELL CYCLE TIME, 363
20.13. CELL MIGRATION, 363
2 1. CYTOTOXICITY, 365 21.1. VIABILITY, TOXICITY, AND SURVIVAL, 365
21.2. IN VITRO LIMITATIONS, 366 21.2.1. PHARMACOKINETICS, 366
21.2.2. /METABOLISM, 366 21.2.3. TISSUE AND SYSTEMIC RESPONSES, 366
21.3. NATURE OF THE ASSAY, 366 21.3.1. VIABILITY, 366
PROTOCOL 21.1. ESTIMATION OF VIABILITY BY DYE EXCLUSION, 367 PROTOCOL
21.2. ESTIMATION OF VIABILITY BY DYE UPTAKE, 367
21.3.2. SURVIVAL, 368 PROTOCOL 21.3. CLONOGENIC ASSAY FOR ATTACHED
CELLS, 368 21.3.3. ASSAYS BASED ON CELL PROLIFERATION, 372
21.3.4. METABOLIC CYTOTOXICITY ASSAYS, 372 21.3.5. MICROTITRATION
ASSAYS, 372 PROTOCOL 21.4. MTT-BASED CYTOTOXICITY ASSAY, 373 21.3.6.
COMPARISON OF MICROTITRATION WITH
CLONOGENIC SURVIVAL, 376 21.3.7. DRUG INTERACTION, 376 21.4.
APPLICATIONS OF CYTOTOXICITY ASSAYS, 377 21.4.1. ANTICANCER DRUG
SCREENING, 377
21.4.2. PREDICTIVE DRUG TESTING FOR TUMORS, 377 21.4.3. TESTING
PHARMACEUTICALS, 377 21.5. GENOTOXICITY, 377
21.5.1. MUTAGENESIS ASSAY BY SISTER CHROMATID EXCHANGE, 377 PROTOCOL
21.5. SISTER CHROMATID EXCHANGE, 378 21.5.2. CARCINOGENICITY, 380 21.6.
INFLAMMATION, 380
22. SPECIALIZED CELLS, 383 22.1. CELL CULTURE OF SPECIALIZED CELLS, 385
22.2. EPITHELIAL CELLS, 385 22.2.1. EPIDERMIS, 385
PROTOCOL 22.1. EPIDERMAL KERATINOCYTES, 387 22.2.2. CORNEA, 390 PROTOCOL
22.2. CORNEAL EPITHELIAL CELLS, 390 22.2.3. BREAST, 391
PROTOCOL 22.3. PREPARATION OF MAMMARY EPITHELIAL CELLS FROM REDUCTION
MAMMOPLASTY SPECIMENS, 392 22.2.4. CERVIX, 393
PROTOCOL 22.4. CERVICAL EPITHELIUM, 393 22.2.5. GASTROINTESTINAL TRACT,
395 PROTOCOL 22.5. ISOLATION AND CULTURE OF COLONIC CRYPTS, 395
22.2.6. LIVER, 397 22.2.7 . HEPATOCYTE PRIMARY CULTURES, 397 PROTOCOL
22.6A. ISOLATION OF RAT HEPATOCYTES, 397 22.2.8. HEPARG HUMAN
HEPATOCYTES, 399
PROTOCOL 22.6B. PURIFICATION OF HEPARG HUMAN HEPATOCYTES, 399 22.2.9.
PANCREAS, 401 PROTOCOL 22. 7. PANCREATIC EPITHELIUM, 401
22.2.10. KIDNEY, 402 PROTOCOL 22.8. KIDNEY EPITHELIUM, 403 22.2.11.
BRONCHIAL AND TRACHEAL EPITHELIUM, 404 PROTOCOL 22.9. BRONCHIAL AND
TRACHEAL EPITHELIUM, 404
22.2.12. ORAL EPITHELIUM, 405 PROTOCOL 22.10. ORAL KERATINOCYTES, 405
22.2.13. PROSTATE, 406 PROTOCOL 22.11. PROSTATIC EPITHELIUM, 407 22.3.
MESENCHYMAL CELLS, 408
22.3.1. CONNECTIVE TISSUE, 408 22.3.2. ADIPOSE TISSUE, 408 PROTOCOL
22.12. PRIMARY CULTURE OF ADIPOSE CELLS, 409
22.3.3. MUSCLE, 410 PROTOCOL 22.13. ISOLATION AND CULTURE OF SMOOTH
MUSCLE CELLS, 410 PROTOCOL 22.14. CULTURE OF MYOBLASTS FROM ADULT
SKELETAL
MUSCLE, 411 PROTOCOL 22.15. SINGLE MYOFIBER CULTURE FROM SKELETAL
MUSCLE, 413 22.3.4. CARTILAGE, 414 PROTOCOL 22.16. CHONDROCYTES IN
ALGINATE BEADS, 414
22.3.5. BONE, 416 PROTOCOL 22.17. OSTEOBLASTS, 417 22.3.6. ENDOTHELIUM,
418 PROTOCOL 22.18. ISOLATION AND CULTURE OF VASCULAR
ENDOTHELIAL CELLS, 419 22.4. NEUROECTODERMAL CELLS, 422 22.4.1. NEURONS,
422 PROTOCOL 22.19. CEREBELLAR GRANULE CELLS, 422
22.4.2. GHAL CELLS, 423 PROTOCOL 22.20. PRIMARY CULTURE OF HUMAN
ASTROCYTES, 424 PROTOCOL 22.21. OLFACTORY ENSHEATHING CELLS, 426
22.4.3. ENDOCRINE CELLS, 428 22.4.4. MELANOCYTES, 429 PROTOCOL 22.22.
CULTURE OF MELANOCYTES, 429 22.5. HEMATOPOIETIC CELLS, 430 22.6. GONADS,
432
IMAGE 10
CONTENTS O* XV
22.6.1.
22.6.2.
OVARY, 432 TESTIS, 432
PROTOCOL 23.11. HEMATOPOIETIC COLONY-FORMING ASSAYS, 461
23. STEM CELLS, GERM CELLS, AND AMNIOCYTES, 433 23.1. STEM CELLS, 433
23.1.1. EMBRYONIC STEM CELLS, 433 23.1.2. DERIVATION OF MOUSE EMBRYONIC
STEM
CELLS, 433
PROTOCOL 23.1. DERIVATION AND PRIMARY CULTURE OF MOUSE EMBRYONIC STEM
CELLS, 434 23.1.3. SUBCULTURE AND PROPAGATION OF MOUSE EMBRYONIC STEM
CELLS, 436 PROTOCOL 23.2. PROPAGATION OF MOUSE EMBRYONIC STEM
CELL LINES, 438 23.1.4. PRIMARY CULTURE OF HUMAN EMBRYONIC STEM CELLS,
439 PROTOCOL 23.3. DERIVATION OF HUMAN EMBRYONIC STEM
CELLS, 440 23.1.5. ^PASSAGING HES CELLS, 440 PROTOCOL 23.4. .^MANUAL
PASSAGE OF HES CELLS, 441 23.1.6. PLURIPOTENT STEM CELLS FROM FISH
EMBRYOS, 442
PROTOCOL 23.5. CELL CULTURES FROM ZEBRAFISH EMBRYOS, 443 23.2. GERM
CELLS, 445 23.3. EXTRAEMBRYONIC CELLS, 445
23.3.1. CULTURE OF AMNIOCYTES, 445 PROTOCOL 23.6. CULTURE OF AMNIOCYTES,
445 23.3.2. CELLS FROM NEONATES AND JUVENILES, 449
23.3.3. MULTIPOTENT STEM CELLS FROM THE ADULT, 449 23.3.4. MSCS FROM
HUMAN BONE MARROW, 450 PROTOCOL 23.7. MSC PRODUCTION FROM HUMAN BONE
MARROW, 450 23.3.5. INDUCED PLURIPOTENT STEM CELLS, 452 PROTOCOL 23.8.
REPROGRAMMING HUMAN DERMAL FIBROBLASTS FOR THE GENERATION OF PLURIPOTENT
STEM
CELLS, 453 PROTOCOL. A. GENERATION OF HUMAN DERMAL FIBROBLAST CELL
LINES, 453 PROTOCOL. B. GENERATION OF HIGH TITERS OF INFECTIVE VIRUS
CODING FOR IPS FACTORS, 453 23.3.6. LONG-TERM BONE MARROW CULTURES FROM
MOUSE, 455 PROTOCOL 23.9. LONG-TERM HEMATOPOIETIC CELL CULTURES
FROM MOUSE BONE MARROW, 456 23.3.7. LONG-TERM CULTURE OF HUMAN PRIMITIVE
HEMOPOIETIC CELLS, 457 PROTOCOL 23.10. HUMAN LONG-TERM
CULTURE-INITIATING
CELL (LTC-IC) ASSAY, 457 23.3.8. HEMATOPOIETIC COLONY-FORMING ASSAYS,
461
R CULTURE OF TUMOR CELLS J 63 24.1. PROBLEMS OF TUMOR CELL CULTURE, 463
24.2. SAMPLING, 464 24.2.1. SELECTION OF REPRESENTATIVE CELLS, 464
24.2.2. PRESERVATION OF TISSUE BY FREEZING, 464 PROTOCOL 24.1. FREEZING
BIOPSIES, 465 24.3. DISAGGREGATION, 465 24.4. PRIMARY CULTURE, 465 24.5.
SELECTIVE CULTURE OF TUMOR CELLS, 466
24.5.1. SELECTIVE MEDIA, 466 24.5.2. CONFLUENT FEEDER LAYERS, 466
PROTOCOL 24.2. GROWTH ON CONFLUENT FEEDER LAYERS, 466 24.5.3. SUSPENSION
CLONING, 467
24.5.4. XENOGRAFTS, 467 24.6. DEVELOPMENT OF CELL LINES, 468 24.6.1.
SUBCULTURE OF PRIMARY TUMOR CULTURES, 468
24.6.2. CONTINUOUS CELL LINES, 469 24.7. CHARACTERIZATION OF TUMOR CELL
CULTURES, 470
24.7.1. HETEROGENEITY OF TUMOR CULTURES, 470 24.7.2. HISTOTYPIC CULTURE,
470 24.8. SPECIFIC TUMOR TYPES, 471
24.8.1. BREAST, 471 PROTOCOL 24.3. CULTURE OF MAMMARY TUMOR CELLS, 472
24.8.2. LUNG, 472
24.8.3. STOMACH, 473 24.8.4. COLON, 473 PROTOCOL 24.4. CULTURE OF
COLORECTAL TUMORS, 473 24.8.5. PANCREAS, 475
24.8.6. OVARY, 475 24.8.7. PROSTATE, 476 24.8.8. BLADDER, 476 24.8.9.
SKIN, 476 24.8.10. CERVIX, 477 24.8.11. GLIOMA, 477 24.8.12.
NEUROBLASTOMA, 478 24.8.13. SEMINOMA, 478 24.8.14. LYMPHOMA AND
LEUKEMIA, 478 PROTOCOL 24.5. ESTABLISHMENT OF CONTINUOUS CELL LINES
FROM LEUKEMIA/LYMPHOMA, 478
25. TBREE-DIMENSIONAL CULTURE, 481
25.1. CELL INTERACTION AND PHENOTYPIC EXPRESSION, 481 25.1.1. EFFECT OF
CELL DENSITY, 481
IMAGE 11
XVI
O CONTENTS
25.1.2. RECIPROCAL INTERACTIONS, 481
25.1.3. CHOICE OF MODELS, 482 25.2. ORGAN CULTURE, 482 25.2.1. GAS AND
NUTRIENT EXCHANGE, 482
25.2.2. STRUCTURAL INTEGRITY, 484 25.2.3. GROWTH AND DIFFERENTIATION,
484 25.2.4. LIMITATIONS OF ORGAN CULTURE, 484 25.2.5. TYPES OF ORGAN
CULTURE, 484 PROTOCOL 25.1. ORGAN CULTURE, 485 25.3. HISTOTYPIC CULTURE,
486
25.3.1. GEL AND SPONGE TECHNIQUES, 486 25.3.2. HOLLOW FIBERS, 487
25.3.3. SPHEROIDS, 487 PROTOCOL 25.2. 3-D CULTURE IN SPHEROIDS, 488
25.3.4. ROTATING CHAMBER SYSTEMS, 489 25.3.5. IMMOBILIZATION OF LIVING
CELLS IN ALGINATE, 490 25.3.6. / FILTER WELL INSERTS, 490 PROTOCOL 25.3.
FILTER WELL INSERTS, 491
25.3.7. CULTURES OF NEURONAL AGGREGATES, 492 PROTOCOL 25,4. NEURONAL
AGGREGATES, 492 25.4. ORGANOTYPIC CULTURE, 493 25.4.1. TISSUE
EQUIVALENTS, 494
25.4.2. TISSUE ENGINEERING, 495 25.5. IMAGING CELLS IN 3-D CONSTRUCTS,
495
26. SCALE-UP AND AUTOMATION, 497 26.1. SCALE-UP IN SUSPENSION, 497
PROTOCOL 26.1. STIRRED 4-LITER BATCH SUSPENSION CULTURE, 498
26.1.1. CONTINUOUS CULTURE, 500 26.1.2. SCALE AND COMPLEXITY, 500
26.1.3. MIXING AND AERATION, 501 26.2. SCALE-UP IN MONOLAYER, 503
26.2.1. MULTISURFACE PROPAGATORS, 504 PROTOCOL 26.2. NUNC CELL FACTORY,
504 26.2.2. ROLLER CULTURE, 505 PROTOCOL 26.3. ROLLER BOTTLE CULTURE,
505
26.2.3. MICROCARRIERS, 506 PROTOCOL 26.4. MICROCARRIERS, 508 26.2.4.
LARGE MICROCARRIERS, 509
26.2.5. PERFUSED MONOLAYER CULTURE, 509 26.3. PROCESS CONTROL, 510 26.4.
AUTOMATION, 513
26.4.1. ROBOTIC CELL CULTURE, 513 26.4.2. HIGH-THROUGHPUT SCREENING, 514
27. SPECIALIZED TECHNIQUES, 517
27.1. LYMPHOCYTE PREPARATION, 517 27.1.1. ISOLATION BY DENSITY, 517
PROTOCOL 27.1. PREPARATION OF LYMPHOCYTES, 517 27.1.2. BLAST
TRANSFORMATION, 518 PROTOCOL 27.2. PHA STIMULATION OF LYMPHOCYTES, 518
27.2. AUTORADIOGRAPHY, 518 PROTOCOL 27.3. MICROAUTORADIOGRAPHY, 519
27.3. TIME-LAPSE RECORDING, 522 PROTOCOL 27.4. TIME-LAPSE VIDEO
RECORDING, 523 27.4. CELL SYNCHRONY, 525
27.4.1. CELL SEPARATION, 525 27.4.2. BLOCKADE, 525 27.5. CULTURE OF
CELLS FROM POIKILOTHERMS, 525 27.5.1. FISH CELLS, 525
27.5.2. INSECT CELLS, 526 PROTOCOL 27.5. PROPAGATION OF INSECT CELLS,
526 27.6. SOMATIC CELL FUSION, 527 27.6.1. CELL HYBRIDIZATION, 527
PROTOCOL 27.6. CELL HYBRIDIZATION, 527 27.6.2. NUCLEAR TRANSFER, 529
27.7. PRODUCTION OF MONOCLONAL ANTIBODIES, 529 PROTOCOL 27.7. PRODUCTION
OF MONOCLONAL ANTIBODIES, 529
28. TRAINING PROGRAMS, 533 28.1. OBJECTIVES, 533 28.2. PREPARATIVE AND
MANIPULATIVE SKILLS, 533 EXERCISE 1 STERILE PIPETTING AND TRANSFER OF
FLUIDS, 536
EXERCISE 2 WASHING AND STERILIZING GLASSWARE, 538 EXERCISE 3 PREPARATION
AND STERILIZATION OF WATER, 538 EXERCISE 4 PREPARATION AND STERILIZATION
OFDULBECCO S PHOSPHATE-BUFFERED SALINE (D-PBS) WITHOUT CA 2+ AND
MG 2+ (D-PBSA), 539 EXERCISE 5 PREPARATION OF PH STANDARDS, 540 EXERCISE
6 PREPARATION OF STOCK MEDIUM FROM POWDER AND STERILIZATION BY
FILTRATION, 541 28.3. BASIC CELL CULTURE TECHNIQUES, 543
EXERCISE 7 OBSERVATION OF CULTURED CELLS, 543 EXERCISE 8 PREPARING
STERILE MEDIUM FOR USE, 545 EXERCISE 9 FEEDING A MONOLAYER CULTURE, 546
EXERCISE 10 PREPARATION OFCOMPLETE MEDIUM FROM LOX
STOCK, 547 EXERCISE 11 COUNTING CELLS BY HEMOCYTOMETER AND ELECTRONIC
COUNTER, 548 EXERCISE 12 SUBCULTURE OF CELLS GROWING
IN SUSPENSION, 551 EXERCISE 13 SUBCULTURE OF CELL LINES GROWING IN
MONOLAYER, 552 EXERCISE 14 STAINING A MONOLAYER CELL CULTURE WITH
GIEMSA, 554 EXERCISE 15 CONSTRUCTION AND ANALYSIS OF GROWTH CURVE, 556
28.4. ADVANCED EXERCISES, 557
EXERCISE 16 CELL LINE CHARACTERIZATION, 558 EXERCISE 17 DETECTION OF
MYCOPLASMA, 559 EXERCISE 18 CRYOPRESERVATION OF CULTURED CELLS, 560
EXERCISE 19 PRIMARY CULTURE, 563
EXERCISE 20 CLONING OF MONOLAYER CELLS, 566 28.5. SPECIALIZED EXERCISES,
568
IMAGE 12
CONTENTS O XVJI
23. PROBLEM SOLVING, 568 29.1. ABNORMAL APPEARANCE OF CELLS, 570 29.2.
SLOW CELL GROWTH, 570 29.2.1. PROBLEMS RESTRICTED TO YOUR OWN
STOCK, 570
29.2.2. PROBLEM MORE GENERAL AND OTHER PEOPLE HAVING DIFFICULTY, 571
29.3. MEDIUM, 572 29.3.1. FORMULATION, PREPARATION, AND
STORAGE, 572
29.3.2. UNSTABLE REAGENTS, 574 29.3.3. PURITY OF MEDIUM CONSTITUENTS,
574 29.4. SUBSTRATES AND CONTAINERS, 575 29.5. MICROBIAL CONTAMINATION,
576 29.5.1. CONFINED TO SINGLE USER, 576 29.5.2. WIDESPREAD, 578 29.5.3.
AIR/SUPPLY AND LAMINAR-FLOW
HOODS, 579
29.5.4. ^SPECIFIC CONTAMINANTS, 580 29.6. CHEMICAL CONTAMINATION, 581
29.6.1. GLASSWARE, 581 29.6.2. PIPETTES, 581
29.6.3. WATER PURIFICATION, 581 29.6.4. CRYOPRESERVATIVES, 581 29.6.5.
POWDERS AND AEROSOLS, 581 29.7. PRIMARY CULTURE, 582
29.7.1. POOR TAKE IN PRIMARY CULTURE, 582 29.7.2. WRONG CELLS SELECTED,
583 29.7.3. CONTAMINATION, 583 29.8. DIFFERENTIATION, 583 29.9. FEEDING,
584
29.9.1. REGULAR MONOLAYERS, 584 29.9.2. CELL CLONING, 584 29.10.
SUBCULTURE, 585 29.10.1. POOR TAKE OR SLOW GROWTH, 585
29.10.2. UNEVEN GROWTH, 585 29.11. CLONING, 586 29.11.1. TOO FEW
COLONIES PER DISH, 586
29.11.2. TOO MANY COLONIES PER DISH, 587 29.11.3. NONRANDOM
DISTRIBUTION, 587 29.12. CROSS-CONTAMINATION AND MISIDENTIFICATION, 588
29.13. CRYOPRESERVATION, 588 29.13.1. POOR RECOVERY, 588 29.13.2.
CHANGED APPEARANCE AFTER CRYOPRESERVATION, 589
29.13.3. LOSS OF STOCK, 590 29.14. CELL COUNTING, 590 29.14.1.
HEMOCYTOMETER, 590
29.14.2. ELECTRONIC COUNTING VIA ORIFICE BY RESISTANCE, 591
30. IN CONCLUSION, 593
APPENDIX I: CALCULATIONS AND PREPARATION OF REAGENTS, 595 CALCULATIONS,
595 CONVERSIONS, 595 PREPARATION OF REAGENTS, 596 APPENDIX II: SOURCES
OF EQUIPMENT AND MATERIALS, 603 APPENDIX III: SUPPLIERS AND OTHER
RESOURCES, 623
APPENDIX IV: GLOSSARY, 633 APPENDIX V: CROSS-CONTAMINATED OR
MISIDENTIFIED CELL LINES, 639 APPENDIX VI: GENERAL TEXTBOOKS AND
RELEVANT
JOURNALS, 661 REFERENCES, 663 INDEX, 111
COMPANION WEBSITE
A COMPANION RESOURCES SITE FOR THIS BOOK IS AVAILABLE AT:
WWW.WILEY.COM/GO/FRESHNEY/CELLCULTURE
|
| 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 | BV036493786 |
| classification_rvk | WC 5100 WX 6600 WX 6603 |
| ctrlnum | (OCoLC)699914973 (DE-599)BVBBV036493786 |
| discipline | Biologie |
| edition | 6. ed. |
| format | Book |
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| id | DE-604.BV036493786 |
| illustrated | Illustrated |
| indexdate | 2024-07-09T22:41:36Z |
| institution | BVB |
| isbn | 9780470528129 |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-020416358 |
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| spelling | Freshney, Robert Ian 1938-2019 Verfasser (DE-588)1055981578 aut Culture of animal cells a manual of basic technique and specialized applications R. Ian Freshney 6. ed. Hoboken, NJ Wiley-Blackwell 2010 XXXI, 732 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Zusatz zum Sachtitel lautete bis zur 5. Aufl. nur "a manual of basic technique" cell culture cabt tissue culture cabt Tierzelle (DE-588)4185509-7 gnd rswk-swf Tiere (DE-588)4060087-7 gnd rswk-swf Gewebekultur (DE-588)4157245-2 gnd rswk-swf Methode (DE-588)4038971-6 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 HEBIS Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=020416358&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis 1\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 and specialized applications cell culture cabt tissue culture cabt Tierzelle (DE-588)4185509-7 gnd Tiere (DE-588)4060087-7 gnd Gewebekultur (DE-588)4157245-2 gnd Methode (DE-588)4038971-6 gnd Zellkultur (DE-588)4067547-6 gnd |
| subject_GND | (DE-588)4185509-7 (DE-588)4060087-7 (DE-588)4157245-2 (DE-588)4038971-6 (DE-588)4067547-6 |
| title | Culture of animal cells a manual of basic technique and specialized applications |
| title_auth | Culture of animal cells a manual of basic technique and specialized applications |
| title_exact_search | Culture of animal cells a manual of basic technique and specialized applications |
| title_full | Culture of animal cells a manual of basic technique and specialized applications R. Ian Freshney |
| title_fullStr | Culture of animal cells a manual of basic technique and specialized applications R. Ian Freshney |
| title_full_unstemmed | Culture of animal cells a manual of basic technique and specialized applications R. Ian Freshney |
| title_short | Culture of animal cells |
| title_sort | culture of animal cells a manual of basic technique and specialized applications |
| title_sub | a manual of basic technique and specialized applications |
| topic | cell culture cabt tissue culture cabt Tierzelle (DE-588)4185509-7 gnd Tiere (DE-588)4060087-7 gnd Gewebekultur (DE-588)4157245-2 gnd Methode (DE-588)4038971-6 gnd Zellkultur (DE-588)4067547-6 gnd |
| topic_facet | cell culture tissue culture Tierzelle Tiere Gewebekultur Methode Zellkultur |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=020416358&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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