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List of protocols | |
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Abbreviations | |
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The cell culture laboratory | |
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Animal cell culture: past, present, and future | |
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The laboratory | |
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Design concepts and layout | |
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Services | |
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Equipping the laboratory | |
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Major items of equipment and instrumentation | |
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Culture plasticware and associated small consumable items | |
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Washing reusable tissue culture equipment | |
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Soaking | |
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Washing | |
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Pipette washing | |
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General care and maintenance of the tissue culture laboratory | |
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Acknowledgements | |
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References | |
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Sterilization | |
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Introduction | |
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What is sterilization? | |
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The importance of sterility in cell culture | |
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The use of antibiotics | |
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Basic principles | |
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Wet heat at up to 100[degree]C | |
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Theory | |
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Pasteurization | |
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100[degree]C | |
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Wet heat above 100[degree]C and autoclaving | |
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Theory | |
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Steam | |
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Types of autoclave | |
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Preparation of the load and operation of the autoclave | |
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Autoclave testing | |
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Dry heat | |
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Incineration | |
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Hot-air ovens | |
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Irradiation | |
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Ultraviolet light | |
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Gamma rays | |
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Chemical sterilization | |
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Fumigation | |
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Liquid disinfectants | |
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Filtration | |
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Filters for bacteria and fungi | |
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Filters for viruses | |
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HEPA filters | |
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Viruses and prions | |
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Virus elimination | |
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Prions | |
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Safety in the laboratory | |
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Good Manufacturing Practice and Good Laboratory Practice | |
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Acknowledgements | |
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References | |
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Culture media | |
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Introduction | |
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Basal media | |
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Types of basal media | |
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Constituents of basal media | |
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Choice of basal medium | |
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Preparation of basal medium | |
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Serum | |
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Why use serum? | |
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Types of serum | |
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Constituents of serum | |
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Potential problems with the use of serum | |
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Sourcing and selection of serum | |
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Serum storage and use | |
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Replacement of serum in media | |
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Reduced-serum media for early-passage normal human cells | |
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Serum-free media (SFM) | |
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Design of SFM | |
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Influence of cell culture systems on the choice of medium | |
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Batch or perfusion cultures | |
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Anchorage-dependent cells | |
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Stirred suspension cultures | |
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High density culture systems | |
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Very low density cultures | |
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In-house medium development and production versus commercial supply | |
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Economic considerations | |
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Development of a dedicated medium | |
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Quality assurance and control | |
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References | |
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Microscopy of living cells | |
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Introduction | |
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The modern light microscope | |
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Basic scheme | |
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Properties of light | |
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Magnification | |
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Resolution | |
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Aberrations and their corrections | |
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Illumination | |
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Maintenance | |
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Microscopic techniques | |
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Bright field | |
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Oblique illumination | |
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Dark field | |
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Phase-contrast | |
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Differential interference contrast (DIC) | |
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Hoffman modulation contrast | |
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Reflection contrast | |
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Varel contrast | |
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Polarizing microscopy | |
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Interference microscopy | |
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Fluorescence microscopy | |
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Specimen preparation | |
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Culture flasks and Petri dishes | |
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Coverslips | |
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Chambers | |
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Interaction with microscopic techniques | |
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Environmental control for microscopic specimens | |
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Recording of microscopic images | |
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Time-lapse imaging | |
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Analysis of microscopic images | |
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Image enhancement by deconvolution | |
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Image processing--extraction of morphometric data | |
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Data analysis--statistical evaluation | |
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Data interpretation--simulation modelling | |
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Acknowledgements | |
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References | |
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Basic cell culture technique and the maintenance of cell lines | |
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Introduction | |
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The terminology of cell and tissue culture | |
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Basic components of the cell culture environment | |
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Culture medium | |
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Physicochemical factors | |
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Stationary versus dynamic media supply | |
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Sterile technique and contamination control | |
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Working within the laminar flow hood | |
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Pipetting and prevention of aerosol formation | |
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Additional considerations for good sterile technique | |
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Prevention of cellular cross-contamination | |
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Cell culture at the open bench | |
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General procedures for the cell culture laboratory | |
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Maintenance of the laboratory | |
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Daily inspection of ongoing cultures | |
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Maintenance of stock cultures | |
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The culture of attached cells | |
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Routine culture substrates | |
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Choice of culture vessels | |
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Artefacts of cell attachment and growth | |
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Use of attachment factors and bio-substrates | |
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Alternative culture substrates | |
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Three-dimensional matrices | |
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Culture of cells on microcarriers | |
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Mass culture systems for adherent cells | |
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The culture of cells in suspension | |
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Non-adherent substrates for small-scale culture | |
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Mass culture of cells in fluid suspension | |
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Micro-encapsulation | |
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In vitro cell growth behaviour | |
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Adaptation to culture | |
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Phases of cell growth | |
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Determinations of cell growth data | |
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Calculation of in vitro age | |
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Multiplication rate and population doubling time | |
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Counting cells in suspension | |
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Counting cells adherent to a substrate | |
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Phases of the cell cycle | |
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Cell generation time by time-lapse photomicrography | |
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Growth fraction | |
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Expressions of culture 'efficiency' | |
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Cryopreservation and retrieval of cells from frozen storage | |
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Purpose of cell banking | |
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Mechanism of cell freezing, and factors that affect viability | |
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Supplies and equipment for cell freezing | |
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Additional comments regarding cryopreservation | |
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Transportation of cells | |
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Transporting frozen cells | |
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Transporting growing cells | |
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Safety in the cell culture laboratory | |
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Potential risks in routine cell culture | |
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Awareness of increased risk associated with human cells | |
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Classification of cell lines as aetiological agents | |
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Precautions in handling pathogenic organisms and human cells | |
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References | |
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Primary culture and the establishment of cell lines | |
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Introduction | |
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Establishment of primary cultures from various sources | |
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Source of material | |
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Isolation of cells | |
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Substrate for attachment | |
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Culture conditions | |
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Evolution of primary cultures | |
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Nomenclature | |
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Subculture | |
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Growth phases | |
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Characteristics of limited lifespan cultures | |
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Lifespan and senescence | |
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Phenotype | |
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Karyotype | |
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Establishment of continuous cell lines | |
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Spontaneous | |
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Chemical transformation | |
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Viral transformation | |
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Properties of continuous cell lines | |
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Aneuploidy | |
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Heterogeneity and instability | |
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Differentiated status | |
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Tumorigenicity | |
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Cell fusion | |
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Methods | |
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Properties of hybrids | |
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Applications | |
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Genetic engineering techniques | |
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Introduction of genes | |
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Oncogene immortalization | |
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Safety considerations | |
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Concluding remarks | |
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Advantages of cell culture over in vivo experimentation | |
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Limitations | |
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Acknowledgements | |
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References | |
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Specific cell types and their requirements | |
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Introduction | |
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General principles | |
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Growth requirements of cells in vitro | |
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Epithelial cells | |
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Mesenchymal cells | |
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Neuro-ectodermal cells | |
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Gonadal cells | |
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Embryonal stem (ES) cells | |
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Leukocytes | |
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Insect cells | |
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Conclusion | |
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Acknowledgements | |
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References | |
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Cloning | |
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Introduction | |
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Development of techniques | |
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Uses of cloning | |
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Limitations of cloning | |
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Special requirements of cells growing at very low densities | |
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Cell cloning procedures | |
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Choice of technique | |
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Methods applicable to both attached and suspension cells | |
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Methods for attached cells | |
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Methods for suspension cells | |
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References | |
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The quality control of cell lines and the prevention, detection, and cure of contamination | |
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Introduction | |
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Obtaining the basic material | |
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Importance of cell culture collections | |
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Resource centres | |
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Cell culture databases | |
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Quarantine and initial handling of cell lines | |
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Accessioning scheme | |
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Laboratory design | |
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Handling of cell lines | |
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Production of cell banks | |
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Microbial quality control | |
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Sources of microbial contamination | |
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Testing for bacteria, yeasts, and other fungi | |
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Testing for mycoplasma | |
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Virus testing | |
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Bovine spongiform encephalopathy | |
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Elimination of contamination | |
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Authentication | |
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Isoenzyme analysis | |
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Cytogenetic analysis | |
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DNA fingerprinting | |
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Regulatory aspects | |
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References | |
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Good Laboratory Practice in the cell culture laboratory | |
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What happens if you fall under a bus? | |
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GLP, an overview | |
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What is GLP and when should it be used? | |
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Planning the study | |
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Performance of the study | |
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Personnel | |
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Methods | |
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Equipment | |
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Materials | |
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Monitoring | |
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Facilities and systems | |
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Study-specific audits | |
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Final report audit | |
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Records | |
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Advice for researchers creating new cell lines | |
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Acknowledgements | |
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References | |
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List of suppliers | |
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Culture collections and other resource centres | |
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Online resources for cell biology | |
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Index | |