Culture of Animal Cells: A Manual of Basic Technique, 4th Edition

by R. Ian Freshney (CRC Beastson Laboratories, Univ. of Glasgow)

• ISBN13: 9780471348894
• ISBN10: 0471348899
• Edition: 4th
• Format: Hardcover
• Copyright: 2000-01-01
• Publisher: Wiley-Liss

Summary

The fourth edition of Culture of Animal Cells: A Manual of Basic Technique offers the most complete training manual of its kind on the fundamental principles and techniques of animal cell culture. Within this volume, indispensable updates reflecting the latest progress in media, specialized techniques, biotechnology, DNA transfer, and tumor culture have been made. This edition has five new chapters expanding on serum-free media, scale-up and biofermentors, molecular techniques, immortalization, and troubleshooting. The advantages of tissue culture go beyond control of the physiochemical environment and physiological conditions as shown in the comprehensive coverage of tissue culture topics, both organ culture and cell culture, provided in this manual. A wide range of essential information from basic to specialized procedures is presented, highlighting advantages and limitations, and illustrating the properties of different types of culture. This crucial reference for cell culture techniques includes: * New Atlas of Cells section in full-color presentation * Extended coverage of molecular techniques, scale-up, and serum-free medium * New chapter on problem solving * Photographs of cell lines, contaminations, and equipment * Clear and concise tables and charts * Educated recommendations on safety issues, ethical consent, and ownership Biomedical researchers in cell biology, cytology, molecular biology, immunology, neuroscience, toxicology, and cancer biology will find Culture of Animal Cells: A Manual of Basic Technique, Fourth Edition to be an invaluable reference.

Table of Contents

Figures	xvii
Color Plates	xxi
Preface	xxiii
Abbreviations	xxv
Introduction	1	(8)
Background	1	(3)
Advantages of Tissue Culture	4	(1)
Control of the Environment	4	(1)
Characterization and Homogeneity of Sample	4	(1)
Economy, Scale, and Mechanization	4	(1)
In Vivo Modeling	5	(1)
Limitations	5	(1)
Expertise	5	(1)
Quantity	5	(1)
Dedifferentiation and Selection	5	(1)
Origin of Cells	5	(1)
Instability	6	(1)
Major Differences In Vitro	6	(1)
Types of Tissue Culture	6	(3)
Biology of Cultured Cells	9	(10)
The Culture Environment	9	(1)
Cell Adhesion	9	(2)
Cell Adhesion Molecules	10	(1)
Cytoskeleton	10	(1)
Cell Proliferation	11	(1)
Cell Cycle	11	(1)
Control of Cell Proliferation	11	(1)
Differentiation	11	(2)
Maintenance of Differentiation	12	(1)
Dedifferentiation	12	(1)
Energy Metabolism	13	(2)
Initiation of the Culture	15	(1)
Evolution of Cell Lines	15	(1)
Senescence	16	(1)
The Development of Continuous Cell Lines	16	(1)
Origin of Cultured Cells	16	(3)
Design and Layout	19	(12)
Planning	19	(1)
Construction and Services	20	(2)
Layout	22	(9)
Sterile Handling Area	23	(1)
Incubation	24	(1)
Hot Room	24	(3)
Service Bench	27	(1)
Preparation	27	(1)
Wash-Up	27	(1)
Storage	28	(3)
Equipment	31	(20)
Requirements of a Tissue Culture Laboratory Essential Equipment	31	(9)
Laminar-Flow Hood	31	(1)
Incubator	32	(2)
CO2 Incubator	34	(1)
Sterilizer	34	(1)
Refrigerators and Freezers	35	(2)
Inverted Microscope	37	(1)
Washing Up	37	(1)
Sterilizing and Drying	38	(1)
Water Purification	38	(1)
Centrifuge	39	(1)
Cryostorage Container	39	(1)
Balance	39	(1)
Hemocytometer	40	(1)
Beneficial Equipment	40	(6)
Cell Counter	40	(1)
Aspiration Pump	40	(1)
pH Meter	41	(1)
Conductivity Meter	41	(1)
Osmometer	41	(1)
Upright Microscope	41	(1)
Dissecting Microscope	41	(1)
Temperature Recording	41	(1)
Magnetic Stirrer	41	(1)
Roller Racks	41	(1)
Fluid Handling	42	(4)
Useful Additional Equipment	46	(2)
Low-Temperature Freezer	46	(1)
Glassware Washing Machine	46	(1)
Video Camera and Monitor	47	(1)
Colony Counter	48	(1)
Controlled-Rate Freezer	48	(1)
Centrifugal Elutriator	48	(1)
Flow Cytometer	48	(1)
Consumable Items	48	(3)
Pipettes	48	(1)
Culture Vessels	49	(1)
Sterile Containers	49	(1)
Syringes and Needles	49	(1)
Sterilization Filters	49	(2)
Aseptic Technique	51	(14)
Objectives of Aseptic Technique	51	(1)
Maintaining Sterility	51	(1)
Elements of Aseptic Environment	52	(3)
Quiet Area	52	(1)
Work Surface	52	(1)
Personal Hygiene	53	(1)
Reagents and Media	53	(1)
Cultures	54	(1)
Sterile Handling	55	(1)
Swabbing	55	(1)
Capping	55	(1)
Flaming	55	(1)
Handling Bottles and Flasks	55	(1)
Pipetting	56	(1)
Pouring	56	(1)
Laminar Flow	56	(2)
Standard Procedure	58	(4)
Working in Laminar Flow	58	(2)
Working on the Open Bench	60	(1)
Petri Dishes and Multiwell Plates	61	(1)
Handling Dishes or Plates	62	(1)
Apparatus and Equipment	62	(3)
Incubators	62	(1)
Boxed Cultures	62	(1)
Gassing with CO2	62	(1)
Training	62	(3)
Safety	65	(12)
Risk Assessment	65	(3)
Standard Operating Procedures	65	(3)
Safety Regulations	68	(1)
General Safety	68	(4)
Operator	68	(1)
Equipment	68	(1)
Glassware and Sharp Items	68	(2)
Chemical Toxicity	70	(1)
Gases	71	(1)
Liquid Nitrogen	71	(1)
Fire	72	(1)
Radiation	72	(1)
Ingestion	72	(1)
Labeled Reagents	72	(1)
High-Energy Sources	72	(1)
Biohazards	73	(4)
Levels of Containment	73	(1)
Human Biopsy Material	73	(2)
Genetic Manipulation	75	(1)
Disposal	76	(1)
Culture Vessels	77	(12)
The Substrate	77	(1)
Attachment and Growth	77	(1)
Substrate Materials	77	(1)
Choice of Culture Vessel	78	(5)
Cell Yield	78	(2)
Suspension Culture	80	(1)
Venting	80	(1)
Sampling and Analysis	81	(1)
Uneven Growth	82	(1)
Cost	82	(1)
Specialized Systems	83	(1)
Permeable Supports	83	(1)
Treated Surfaces	83	(6)
Matrix Coating	83	(1)
Preparation of ECM	84	(1)
Feeder Layers	85	(1)
Three-dimensional Matrices	85	(1)
Alternative Artificial Substrates	85	(1)
Nonadhesive Substrates	86	(1)
Liquid-Gel or Liquid-Liquid Interfaces	86	(3)
Media	89	(15)
Development of Media	89	(1)
Physicochemical Properties	89	(4)
pH	89	(1)
Preparations of pH Standards	90	(1)
CO2 and Bicarbonate	90	(1)
Buffering	91	(1)
Oxygen	91	(1)
Osmolality	92	(1)
Temperature	92	(1)
Viscosity	93	(1)
Surface Tension and Foaming	93	(1)
Balanced Salt Solutions	93	(1)
Complete Media	94	(5)
Amino Acids	94	(1)
Vitamins	95	(1)
Salts	95	(1)
Glucose	95	(1)
Organic Supplements	95	(1)
Hormones and Growth Factors	95	(1)
Antibiotics	95	(4)
Serum	99	(2)
Protein	99	(1)
Growth Factors	99	(1)
Hormones	100	(1)
Nutrients and Metabolites	101	(1)
Lipids	101	(1)
Minerals	101	(1)
Inhibitors	101	(1)
Selection of Medium and Serum	101	(2)
Batch Reservation	103	(1)
Testing Serum	103	(1)
Heat Inactivation	103	(1)
Other Supplements	103	(1)
Embryo Extract	104	(1)
Conditioned Medium	104	(1)
Serum-Free Media	104	(17)
Disadvantages of Serum	105	(5)
Advantages of Serum-Free Media	110	(1)
Selective Media	110	(1)
Regulation of Proliferation and Differentiation	111	(1)
Disadvantages of Serum-Free Media	111	(1)
Replacement of Serum	111	(5)
Subculture	111	(1)
Hormones	111	(5)
Growth Factors	116	(1)
Nutrients in Serum	116	(1)
Proteins and Polyamines	116	(1)
Matrix	116	(1)
Selection of Serum-Free Medium	116	(1)
Commercially Available Serum-Free Media	117	(1)
Serum Substitutes	117	(1)
Development of Serum-Free Medium	117	(3)
Preparation of Serum-Free Medium	120	(1)
Conclusions	120	(1)
Preparation and Sterilization	121	(28)
Apparatus	121	(9)
Glassware	121	(1)
Preparation and Sterilization of Glassware	122	(3)
Pipettes	125	(1)
Preparation and Sterilization of Pipettes	125	(2)
Screw Caps	127	(1)
Preparation and Sterilization of Screw Caps	128	(1)
Selection of Detergent	128	(1)
Miscellaneous Equipment	129	(1)
Reusable Sterilizing Filters	130	(1)
Sterilizing Filter Assemblies	130	(1)
Reagents and Media	130	(16)
Water	131	(1)
Balanced Salt Solutions	132	(1)
Preparation of BSS	132	(1)
Media	133	(1)
Preparation of Medium from 1x Stock	133	(1)
Preparation of Medium from 10x Concentrate	134	(2)
Powdered Media	136	(1)
Preparation of Medium from Powder	136	(1)
Customized Medium	137	(1)
Preparation of Customized Medium	137	(2)
Sterilization	139	(1)
Sterile Filtration	139	(1)
Sterile Filtration with Syringe-tip Filter	140	(1)
Sterile Filtration with Filter Flask	141	(1)
Sterile Filtration with Small In-line Filter	142	(1)
Sterile Filtration with Large In-line Filter	142	(1)
Serum	143	(1)
Collection and Sterilization of Serum	143	(1)
Serum Dialysis	144	(2)
Preparation and Sterilization of Other Reagents	146	(1)
Control, Testing, and Storage of Media	146	(3)
Quality Control	146	(1)
Sterility Testing	146	(1)
Culture Testing	146	(1)
Storage	147	(2)
Primary Culture	149	(28)
Types of Primary Cell Culture	149	(1)
Isolation of the Tissue	149	(8)
Mouse Embryo Cell Culture	150	(1)
Isolation of Mouse Embryos	150	(2)
Chick Embryo Cell Culture	152	(1)
Isolation of Chick Embryos	152	(1)
Human Biopsy Material	153	(4)
Human Biopsies	157	(1)
Primary Culture	157	(20)
Primary Explant	157	(1)
Primary Explants	158	(1)
Enzymatic Disaggregation	159	(2)
Warm Trypsin	161	(2)
Trypsinization with Cold Preexposure	163	(1)
Cold Trypsin	163	(2)
Chick Embryo Organ Rudiments	165	(1)
Chick Embryo Organ Rudiments	165	(4)
Other Enzymatic Procedures	169	(1)
Collagenase	169	(1)
Disaggregation in Collagenase	169	(2)
Mechanical Disaggregation	171	(2)
Mechanical Disaggregation by Sieving	173	(1)
Separation of Viable and Nonviable Cells	173	(1)
Enrichment of Viable Cells	173	(2)
Primary Records	175	(2)
Cell Lines	177	(18)
Nomenclature	177	(1)
Subculture and Propagation	177	(3)
Immortalization of Cell Lines	180	(1)
Cell Line Designations	180	(1)
Selection of Cell Line	181	(1)
Routine Maintenance	181	(14)
Cell Morphology	181	(1)
Replacement of Medium	181	(1)
Volume, Depth, and Surface Area	181	(1)
Changing the Medium or Feeding	182	(1)
Holding Medium	183	(1)
Subculture	183	(1)
Criteria for Subculture	183	(1)
Subculture	184	(4)
Split Ratios and Growth Cycle	188	(1)
Cell Concentration at Subculture	188	(1)
Propagation in Suspension	189	(1)
Subculture of Suspension Culture	189	(1)
Subculture in Suspension	189	(2)
Use of Antibiotics	191	(2)
Maintenance Records	193	(2)
Cloning and Selection	195	(20)
Cloning	195	(3)
Dilution Cloning	196	(2)
Stimulation of Plating Efficiency	198	(2)
Improving Clonal Growth	198	(1)
Conditioned Medium	199	(1)
Preparation of Conditioned Medium	199	(1)
Feeder Layers	199	(1)
Preparation of Feeder Layers	200	(1)
Suspension Cloning	200	(4)
Cloning in Agar	200	(2)
Cloning in Methocel	202	(2)
Isolation of Clones	204	(3)
Isolation of Clones with Cloning Rings	206	(1)
Isolating Cell Colonies by Irradiation	206	(1)
Other Isolation Techniques for Monolayer Clones	206	(1)
Suspension Clones	206	(1)
Isolation of Suspension Clones	206	(1)
Replica Plating	207	(1)
Selective Inhibitors	207	(1)
Isolation of Genetic Variants	208	(3)
Methotrexate Resistance and DHFR Amplification	208	(3)
Interaction with Susbstrate	211	(4)
Selective Adhesion	211	(1)
Selective Detachment	211	(1)
Nature of Substrate	211	(1)
Selective Feeder Layers	212	(1)
Semisolid Media	213	(2)
Cell Separation	215	(14)
Cell Density and Isopyknic Sedimentation	215	(3)
Cell Separation by Density Gradient	215	(3)
Variations	218	(1)
Antibody-Based Techniques	218	(1)
Immune Panning	218	(1)
Magnetic Sorting	219	(3)
Magnetic-Activated Cell Sorting (MACS)	219	(3)
Cell Size and Sedimentation Velocity	222	(1)
Centrifugal Elutriation	222	(1)
Fluorescence-Activated Cell Sorting	223	(2)
Other Techniques	225	(1)
Beginner's Approach to Cell Separation	226	(3)
Characterization	229	(30)
The Need for Characterization	229	(2)
Species Identification	230	(1)
Lineage or Tissue Markers	230	(1)
Unique Markers	231	(1)
Transformation	231	(1)
Morphology	231	(10)
Microscopy	234	(1)
Using an Inverted Microscope	234	(1)
Staining	234	(1)
Staining with Giemsa	235	(1)
Staining with Crystal Violet	236	(1)
Culture Vessels for Cytology: Monolayer Cultures	236	(1)
Preparation of Suspension Cultures for Cytology	236	(1)
Smear Preparation	236	(1)
Cytocentrifuge	236	(2)
Filtration Cytology	238	(1)
Photography	239	(1)
Microscope Photography on Film	239	(1)
Electronic Image Recording	240	(1)
Electronic Images from Microscope	241	(1)
Chromosome Analysis	241	(4)
Chromosome Preparations	242	(1)
Chromosome Banding	242	(1)
Chromosome Analysis	243	(2)
Chromosome Painting	245	(1)
DNA Content	245	(4)
DNA Hybridization	246	(1)
DNA Fingerprinting	246	(1)
Multilocus DNA Fingerprinting of Cell Lines	247	(2)
RNA and Protein	249	(2)
Enzyme Activity	251	(3)
Isoenzymes	251	(1)
Isoenzyme Analysis	252	(2)
Antigenic Markers	254	(3)
Indirect Immunofluorescence	256	(1)
Differentiation	257	(1)
Authentication	257	(2)
Differentiation	259	(10)
Expression of In Vivo Phenotype	259	(1)
Dedifferentiation	259	(1)
Stages of Commitment and Differentiation	259	(1)
Proliferation and Differentiation	260	(1)
Commitment and Lineage	260	(1)
Markers of Differentiation	261	(1)
Induction of Differentiation	262	(4)
Soluble Inducers	262	(1)
Cell Interaction	262	(2)
Paracrine Growth Factors	264	(1)
Negatively Acting Paracrine Factors	265	(1)
Cell-Matrix Interactions	265	(1)
Polarity and Cell Shape	266	(1)
Differentiation and Malignancy	266	(1)
Practical Aspects	266	(3)
Transformation	269	(16)
Role in Cell Line Characterization	269	(1)
What is Transformation?	269	(1)
Genetic Instability	269	(2)
Chromosomal Aberrations	271	(1)
DNA Content	271	(1)
Immortalization	271	(6)
Control of Senescence	272	(1)
Immortalization with Viral Genes	273	(1)
Immortalization of Human Fibroblasts	274	(1)
Fibroblast Immortalization	274	(3)
Telomerase-induced Immortalization	277	(1)
Transgenic Mouse	277	(1)
Aberrant Growth Control	277	(3)
Anchorage Independence	277	(1)
Contact Inhibition	278	(1)
Density Limitation of Cell Proliferation	279	(1)
Serum Dependence	279	(1)
Tumorigenicity	280	(5)
Malignancy	280	(1)
Tumorigenesis	281	(1)
Invasiveness	281	(1)
Angiogenesis	282	(1)
Plasminogen Activator	283	(2)
Contamination	285	(12)
Sources of Contamination	285	(4)
Operator Technique	285	(1)
Environment	285	(3)
Use and Maintenance of Laminar-flow Hood Humid Incubators	288	(1)
Cleaning Incubators	288	(1)
Cold Stores	289	(1)
Sterile Materials	289	(1)
Imported Cell Lines and Biopsies	289	(1)
Quarantine	289	(1)
Types of Microbial Contamination	289	(1)
Monitoring for Contamination	289	(5)
Visible Microbial Contamination	289	(1)
Mycoplasma	290	(2)
Fluorescence Detection of Mycoplasma	292	(2)
Viral Contamination	294	(1)
Eradication of Contamination	294	(1)
Bacteria, Fungi, and Yeasts	294	(1)
Eradication of Microbial Contamination	294	(1)
Eradication of Mycoplasma	294	(1)
Eradication of Viral Contamination	295	(1)
Persistent Contamination	295	(1)
Cross-Contamination	295	(1)
Conclusions	296	(1)
Cryopreservation	297	(12)
Need for Cryopreservation	297	(1)
Rationale for Freezing	297	(1)
Preservation	297	(10)
Selection of Cell Line	297	(1)
Standardization of Culture Conditions	297	(1)
Stages of Cryopreservation	298	(1)
Freezing Cells	299	(3)
Cooling Rate	302	(1)
Cryofreezers	302	(1)
Freezer Records	303	(1)
Thawing Frozen Cells	303	(4)
Serial Replacement	307	(1)
Cell Banks	307	(1)
Transporting Cells	308	(1)
Frozen Ampules	308	(1)
Living Cultures	308	(1)
Quantitation	309	(20)
Cell Counting	309	(5)
Hemocytometer	309	(1)
Cell Counting by Hemocytometer	309	(3)
Electronic Counting	312	(1)
Electronic Cell Counting	312	(1)
Cell Sizing	313	(1)
Stained Monolayers	313	(1)
Cell Weight	314	(1)
DNA Content	314	(1)
DNA Estimation by Hoechst 33258	314	(1)
Protein	314	(1)
Solubilization of Sample	315	(1)
Protein Estimation by Bradford Method	315	(1)
Rates of Synthesis	315	(2)
DNA Synthesis	315	(1)
DNA Synthesis	315	(1)
Protein Synthesis	316	(1)
Protein Synthesis	316	(1)
Preparation of Samples for Enzyme Assay and Immunoassay	317	(1)
Cytometry	317	(1)
In situ Labeling	317	(1)
Flow Cytometry	318	(1)
Replicate Sampling	318	(1)
Data Acquisition	318	(1)
Data Analysis	318	(1)
Cell Proliferation	319	(4)
Experimental Design	319	(1)
Growth Cycle	319	(1)
Growth Curve, Monolayer	319	(2)
Suspension Cultures	321	(1)
Growth Curve, Suspension	321	(1)
Analysis of the Growth Cycle	322	(1)
Plating Efficiency	323	(2)
Plating Efficiency	324	(1)
Automatic Colony Counting	325	(1)
Labeling Index	325	(2)
Labeling Index with [3H]-thymidine	325	(2)
Growth Fraction	327	(1)
Determination of Growth Fraction	327	(1)
Mitotic Index	327	(1)
Division Index	327	(1)
Cell Cycle Time (Generation Time)	327	(1)
Cell Migration	328	(1)
Cytotoxicity	329	(16)
Introduction	329	(1)
In Vitro Limitations	330	(1)
Nature of the Assay	330	(10)
Viability	330	(1)
Estimation of Viability by Dye Exclusion	331	(1)
Estimation of Viability by Dye Uptake	331	(1)
Survival	332	(1)
Clonogenic Assay	332	(3)
Cell Proliferation Assays	335	(1)
Metabolic Assays	335	(1)
Microtitration Assays	335	(1)
MTT-Based Cytotoxicity Assay	336	(2)
Comparison of Microtitration with Cloning	338	(2)
Drug Interaction	340	(1)
Anticancer Drug Screening	340	(1)
Predictive Testing	340	(1)
Transformation	340	(3)
Sister Chromatid Exchange	341	(2)
Carcinogenicity	343	(1)
Inflammation	343	(2)
Specialized Cells	345	(40)
Epithelial Cells	345	(19)
Epidermis	346	(1)
Epidermal Keratinocytes	347	(2)
Cornea	349	(1)
Corneal Epithelial Cells	350	(1)
Breast	351	(1)
Mammary Epithelium	351	(1)
Cervix	352	(1)
Cervical Epithelium	352	(3)
Gastrointestinal Tract	355	(1)
Colorectal Epithelium	355	(2)
Liver	357	(1)
Isolation of Rat Hepatocytes	357	(1)
Pancreas	358	(1)
Pancreatic Epithelium	358	(1)
Kidney	359	(1)
Kidney Epithelium	360	(1)
Bronchial and Tracheal Epithelium	361	(1)
Bronchial and Tracheal Epithelium	361	(1)
Prostate	362	(1)
Prostatic Epithelium	362	(2)
Mensenchymal Cells	364	(9)
Connective Tissue	364	(1)
Adipose Tissue	364	(1)
Primary Culture of Adipose Cells	365	(1)
Muscle	366	(1)
Skeletal Muscle	366	(1)
Cartilage	367	(1)
Chondrocytes in Alginate Beads	368	(2)
Bone	370	(1)
Osteoblasts	370	(2)
Endothelium	372	(1)
Vascular Endothelial Cells	372	(1)
Neuroectodermal Cells	373	(7)
Neurons	373	(1)
Cerebellar Granule Cells	373	(1)
Glial Cells	374	(2)
Olfactory Bulb Ensheathing Cells	376	(2)
Endocrine Cells	378	(1)
Melanocytes	378	(1)
Melanocytes	378	(2)
Hematopoietic Cells	380	(4)
Long-Term Bone Marrow Cultures	381	(1)
Long-term Hematopoietic Cell Cultures from Bone Marrow	381	(1)
Hematopoietic Colony-Forming Assays	382	(1)
Hematopoietic Colony-forming Assays	382	(2)
Gonads	384	(1)
Germ Cells	384	(1)
Tumor Cells	385	(10)
Sampling	386	(1)
Disaggregation	387	(1)
Primary Culture	387	(1)
Characterization	387	(1)
Development of Cell Lines	388	(1)
Continuous Cell Lines	388	(1)
Selective Culture	388	(4)
Selective Media	389	(1)
Confluent Feeder Layers	389	(1)
Growth on Confluent Feeder Layers	390	(1)
Suspension Cloning	391	(1)
Histotypic Culture	391	(1)
Spheroids	391	(1)
Xenografts	391	(1)
Preservation of Tissue by Freezing	391	(1)
Freezing Biopsies	391	(1)
Specific Tumor Types	392	(3)
Breast	392	(1)
Lung	392	(1)
Colon	392	(1)
Pancreas	392	(1)
Ovary	392	(1)
Prostate	393	(1)
Skin	393	(1)
Cervix	393	(1)
Neuroblastoma	393	(1)
Seminoma	393	(2)
Organotypic Culture	395	(12)
Cell Interaction and Phenotypic Expression	395	(1)
Reciprocal Interactions	395	(1)
Choice of Models	396	(1)
Organ Culture	396	(3)
Gas and Nutrient Exchange	396	(1)
Structural Integrity	396	(1)
Growth and Differentiation	397	(1)
Limitations of Organ Culture	397	(1)
Types of Organ Culture	397	(1)
Organ Culture	397	(2)
Histotypic Culture	399	(4)
Gel and Sponge Techniques	399	(1)
Hollow Fibers	399	(1)
Spheroids	399	(1)
Spheroids	400	(2)
Immobilization of Living Cells in Alginate	402	(1)
Alginate Encapsulation	402	(1)
Filter-Well Inserts	403	(4)
Filter-Well Inserts	404	(1)
Cultures of Neuronal Aggregates	405
Neuronal Aggregates	405z	(406)
Organotypic Culture	406	(1)
Scale-Up	407	(16)
Scale-up Suspension	407
Stirred 4-liter Batch Suspension Culture	407	(2)
Continuous Culture	409	(1)
Scale and Complexity	410	(1)
Mixing and Aeration	410
Scale-up Monolayer	000	(423)
Multisurface Propagators	412	(1)
Nunclon Cell Factory	413	(1)
Multiarray Disks, Spirals, and Tubes	414	(1)
Roller Culture	414	(1)
Roller Bottle Culture	414	(2)
Microcarriers	416	(1)
Microcarriers	417	(2)
Perfused Monolayer Culture	419	(1)
Monitoring Growth	420	(3)
Specialized Techniques	423	(20)
Lymphocyte Preparation	423	(1)
Preparation of Lymphocytes	423	(1)
Blast Transformation	424	(1)
PHA Stimulation of Lymphocytes	424	(1)
Autoradiography	424	(5)
Microautoradiography	425	(4)
Time-Lapse Recording	429	(2)
Time-Lapse Video	430	(1)
Confocal Microscopy	431	(1)
Cell Synchrony	431	(1)
Cell Separation	431	(1)
Blockade	431	(1)
Culture of Amniocytes	432	(5)
Culture of Amniocytes	432	(5)
Culture of Cells from Poikilotherms	437	(6)
Fish Cells	437	(1)
Reagents and Media for Protocols 26.6--26.8	438	(1)
Fibroblast Feeder Layers from Zebrafish Embryos	439	(1)
Primary Cultures from Zebrafish Embryos	439	(1)
Cell Lines from Zebrafish Embryos	440	(1)
Insect Cells	440	(1)
Propagation of Insect Cells	440	(3)
Molecular Techniques	443	(20)
Molecular Biology in Cell Culture	443	(1)
In Situ Molecular Hybridization	443	(6)
Analysis of RNA Gene Expression by in Situ Hybridization	443	(1)
Autoradiographic in Situ Hybridization	443	(4)
Fluorescence in Situ Hybridization in the Analysis of Genes and Chromosomes	447	(1)
FISH Using Single-Copy Genomic Probes and Chromosome Painting	447	(2)
Somatic Cell Fusion	449	(3)
Cell Hybridization	449	(2)
Selection of Hybrid Clones	451	(1)
Nuclear Transfer	451	(1)
Monochromosomal Transfer	451	(1)
Production of Monoclonal Antibodies	452	(3)
Production of Monoclonal Antibodies	452	(3)
DNA Transfer	455	(8)
Coprecipitation with Calcium Phosphate	456	(1)
Stable DNA Transfection with Calcium Phosphate	456	(1)
Lipofection	457	(1)
Transient Transfection by Lipofection	457	(1)
Electroporation	458	(1)
Stable Transfection by Electroporation	459	(1)
Other DNA Transfer Methods	460	(1)
Reporter Genes	460	(1)
In Situ Staining for &beat;-galactosidase	461	(1)
Chloramphenicol Acetyltransferase (CAT) Assay	461	(2)
Problem Solving	463	(12)
Slow Cell Growth	463	(2)
Is the problem restricted to your own stocks or are other people having similar problems?	463	(1)
If the problem is more general and other researchers are having difficulty as well, check shared facilities and reagents	464	(1)
Have any changes occurred in the laboratory?	464	(1)
Medium	464	(1)
Choice of Medium	465	(1)
Selection of the correct medium is critical	465	(1)
Unstable Reagents	466	(1)
Glutamine	466	(1)
Serum	466	(1)
Other Constituents	466	(1)
Trypsin	466	(1)
Purity of Constituents	466	(1)
Plastics	466	(1)
Glassware	466	(1)
Wash-Up	466	(1)
Contamination	467	(2)
Single User	467	(1)
Widespread	467	(1)
Identification of Contamination	468	(1)
Chemical Contamination	469	(1)
Primary Culture	469	(1)
Poor Take in Primary Culture	469	(1)
Wrong Cells	470	(1)
Contamination	470	(1)
Cloning	470	(1)
Poor Plating Efficiency	470	(1)
Diffuse Colonies	471	(1)
Too Many Colonies per Dish	471	(1)
Nonrandom Distribution	471	(1)
Nonadherent Cells	471	(1)
Differentiation	471	(1)
Cells Do Not Differentiate	471	(1)
Loss of Product Formation	471	(1)
Feeding	471	(1)
Clones	471	(1)
Subculture	471	(1)
Cell Cycle Phase at Subculture	472	(1)
Senescence	472	(1)
Medium	472	(1)
Uneven Growth	472	(1)
Cross-Contamination	472	(1)
Symptoms	472	(1)
Prophylaxis	472	(1)
Cure	472	(1)
Cryopreservation	472	(1)
Poor Recovery	472	(1)
Changed Appearance After Cryopreservation	473	(1)
Contamination	473	(1)
Loss of Stock	473	(1)
Cell Counting	473	(1)
Hemocytometer	473	(1)
Electronic	473	(1)
Viability	474	(1)
Morphological Appearance	474	(1)
Testing Viability	474	(1)
Cytotoxicity	474	(1)
In Conclusion	475	(2)
Reagent Appendix	477	(6)
Trade Index	483	(1)
Sources of Materials	483	(5)
Suppliers and Other Resources	488	(29)
Glossary	517	(6)
References	523	(38)
General Textbooks	561	(2)
Useful Journals	563	(2)
Index	565