Cell Biology

Cell Biology

A Laboratory Handbook

1st Edition - January 1, 1994

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  • Editor: Julio E Celis
  • eBook ISBN: 9781483282459

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Cell Biology: A Laboratory Handbook, Volume 3 is a handbook on cell biology and covers topics ranging from transfer of macromolecules and small molecules to cloning of embryos, transgenics, and gene targeting. Cell-free extracts, permeabilized cell systems, and expression systems are also discussed, along with proteins. Comprised of 58 chapters, this volume begins with a detailed account of microinjection of RNA, DNA, and proteins into somatic cells, followed by an analysis of computer-automated capillary microinjection of macromolecules into living cells. The reader is then introduced to syringe loading as a method for inserting macromolecules into cells in suspension; electroporation of cells; and the use of liposomes in drug targeting. Subsequent chapters focus on the cloning of rabbit embryos by nuclear transplantation; gene targeting by homologous recombination in embryonic stem cells; production and isolation of recombinant viruses; and gel electrophoresis. This book will be of interest to geneticists and molecular biologists.

Table of Contents

  • Part 1: Tissue Culture and Associated Techniques

    Section A: General Techniques

    General Procedures for Tissue Culture

    Development of Serum-Free Media and Methods for Optimization of Nutrient Composition

    Testing Cell Cultures for Microbial and Viral Contaminants

    Section B: Primary Cultures from Embryonic Tissues

    Primary and Extended Culture of Embryonic Mouse Cells: Establishment of a Novel Cell Culture Model of Apoptosis and Neural Differentiation

    Tissue Culture of Embryonic Stem Cells

    Isolation and Culture of Germ Cells from the Mouse Embryo

    Section C: Cultures of Specific Cell Types

    Epithelial Cells

    Cultivation of Human Epidermal Keratinocytes with a 3T3 Feeder Layer

    Growth of Human Keratinocytes in Serum-Free Medium

    Isolation of Hepatocytes

    Isolation and Culture of Oval Cells from Carcinogen-Treated Rats

    In Vitro Culture of Mouse Fetal Choroid Plexus Epithelial Cells

    Isolation and Culture of Type II Pulmonary Epithelial Cells

    Mesenchymal Cells

    Maintenance of Human Diploid Fibroblast-Like Cells in Culture

    Isolation of Osteoclasts and Osteoclast Plasma Membranes

    Culturing of Human Umbilical Vein and Dermal Microvascular Endothelial Cells

    Neuroectodermal Cells

    Isolation and Proliferation of Adult Mammalian Central Nervous System Stem Cells

    Hemopoietic Cells

    Clonal Cultures in Vitro for Hemopoietic Cells Using Semisolid Agar Medium


    Properties of Isolated Sertoli Cells

    Culture of Ovarian Granulosa Cells: Calcium Imaging at the Single-Cell Level

    Section D: Cell Separation Techniques

    Isolation of Peripheral Blood Mononuclear Cells and Identification of Human Lymphocyte Subpopulations by Multiparameter Flow Cytometry

    Purification of Functionally Active Epidermal Langerhans Cells Using Immunomagnetic Beads

    Section E: Model Systems to Study Differentiation

    Nonterminal and Terminal Adipocyte Differentiation of Murine 3T3 T Mesenchymal Stem Cells

    Cell Systems for ex Vivo Studies of Myogenesis: A Protocol for the Isolation of Stable Muscle Cell Populations from Newborn to Adult Mice

    Induction of Cell Differentiation in Human HL-60 Promyelocyte Leukemia Cells: Quantitation of a Myeloid Specific Antigen, MRP-8/MRP-14 Protein Complex

    Differentiation of Murine Erythroleukemia Cells (Friend Cells)

    Cultured PC 12 Cells: A Model for Neuronal Function and Differentiation

    Growing Madin-Darby Canine Kidney Cells for Studying Epithelial Cell Biology

    In Vitro Studies of Epithelium-to-Mesenchyme Transitions

    Section F: Immortalization of Cells

    Inducible Immortalization of Cells from Transgenic Mice Expressing Simian Virus 40 under lac Operon Control

    Immortalization of Rat Ventral Prostate Epithelial Cells Using Simian Virus 40 T Antigen

    Section G: Cell Cycle Analysis

    Cell Cycle Analysis by Flow Cytometry

    Preparation of Synchronous Populations of Mammalian Cells in Specific Phases of the Cell Cycle by Centrifugal Elutriation

    Synchronization of Normal Diploid and Transformed Mammalian Cells

    Synchronization of Transformed Human Amnion Cells by Mitotic Detachment

    Stimulation of DNA Synthesis in Quiescent 3T3 Cells

    Section H: Cytotoxic Assays

    Quantitative Determination of Compound Cytotoxicity in Proliferating Cells: Monitoring DNA Synthesis by [3H]Thymidine Incorporation

    Section I: Senescence, Programmed Cell Death, and Others

    Serial Propagation of Human Fibroblasts for the Study of Aging at the Cellular Level

    Morphological Criteria for Identifying Apoptosis

    Use of the Terminal Transferase DNA Labeling Reaction for the Biochemical and in Situ Analysis of Apoptosis

    Growth and Induction of Metastasis of Mammary Epithelial Cells

    Measurement of Cell-Cell and Cell-Extracellular Matrix Interactions: A Quantitative Cell Attachment Assay

    Section J: Electrophysiological Methods

    Patch-Clamp Recording

    Section K: Histocultures

    Three-Dimensional Sponge-Gel Matrix Histoculture: Methods and Applications

    Section L: Other Cell Types


    Primary Culture of Drosophila Embryo Cells

    Caenorhabditis elegans

    Laboratory Cultivation of Caenorhabditis elegans and Other Free-Living Nematodes


    Cultivation of Tetrahymena Cells

    Acanthamoeba castellanii: A Model System for Correlative Biochemical and Cell Biological Studies


    Cell Biological, Molecular Genetic, and Biochemical Methods to Examine Dictyostelium

    Large-Scale Culture of Physarum: A Simple Method for Growing Several Hundred Grams of Plasmodia


    Induction of Regeneration-Competent Monocot Callus

    Isolation, Culture, and Plant Regeneration from Protoplasts

    Part 2: Viruses

    Propagation and Purification of Polyoma and Simian Virus 40

    Construction and Propagation of Human Adenovirus Vectors

    Tissue Culture Techniques for the Study of Human Papilloma Viruses

    Growth and Purification of Murine Leukemia Virus

    Part 3: Organelles, Cellular Structures, Macromolecules, and Functional Assays

    Purification of Rat Liver Golgi Stacks

    Preparation and Purification of Post-Golgi Transport Vesicles from Perforated Madin-Darby Canine Kidney Cells

    Purification of Clathrin-Coated Vesicles from Bovine Brain, Liver, and Adrenal Gland

    Functional Identification of Membranes Derived from the Rough Endoplasmic Reticulum of Yeast

    Isolation of Yeast Mitochondria and Study of Mitochondrial Protein Translation

    Inclusion of Proteins into Isolated Mitochondrial Outer Membrane Vesicles

    Isolation of Peroxisomes

    Purification of Secretory Granules from PC 12 Cells

    Preparation of Synaptic Vesicles from Mammalian Brain

    Purification and Reconstitution of the Ca2+ -ATPase of Red Blood Cells

    Isolation of Focal Adhesions from Cultured Cells

    Isolation of Laminins from Tumor Sources and from Normal Tissues

    Isolation of Centrosomes from Cultured Animal Cells

    Preparation of Yeast Spindle Pole Bodies

    Preparation of Nuclei and Nuclear Envelopes, Identification of an Integral Membrane Protein Unique to the Nuclear Envelope

    Preparation of Cytoplasts and Karyoplasts from HeLa Cell Monolayers

    Isolation and Visualization of the Nuclear Matrix, the Nonchromatin Structure of the Nucleus

    Preparation of U Small Nuclear Ribonucleoprotein Particles

    Rapid Preparation of hnRNP Core Proteins and Stepwise Assembly of hnRNP Particles in Vitro

    Preparation of Ribosomes and Ribosomal Proteins from Cultured Cells

    Preparation of Proteasomes

    Small-Scale Preparation of Nuclear Extracts from Mammalian Cells

    Purification of DNA Using Guanidine Thiocyanate and Isobutyl Alcohol Fractionation

    Single-Step Method of Total RNA Isolation by Acid Guanidine-Phenol Extraction

    Part 4: Microscopy Techniques

    Section A: Light Microscopy

    Phase-Contrast, Nomarski (Differential-Interference) Contrast, and Dark- Field Microscopy: Black and White and Color Photomicrography

    Reflection Interference Microscopy

    Using Interference Microscopy to Study Cell Behavior

    Section B: Fluorescence Microscopy

    Fluorescence Imaging in Living Cells

    Practical Laser-Scanning Confocal Light Microscopy: Obtaining Optimal Performance from Your Instrument

    Caged Fluorescent Probes for Monitoring Cytoskeleton Dynamics

    Section C: Video Microscopy

    Video-Enhanced Contrast Microscopy

    Section D: Confocal Microscopy

    Confocal Microscopy of Polarized MDCK Epithelial Cells

    Real-Time Confocal Microscopy and Cell Biology

    Section E: Electron Microscopy

    Fixation of Cells and Tissues for Transmission Electron Microscopy

    Embedding of Cells and Tissues for Ultrastructural and Immunocytochemical Analysis

    Negative Staining

    Whole-Mount Electron Microscopy of the Cytoskeleton: Negative Staining Methods

    Glycerol Spraying/Low-Angle Rotary Metal Shadowing

    Rapid Freezing of Biological Specimens for Freeze-Fracture and Deep Etching

    Freeze Fracture and Freeze Etching

    Use of Ultrathin Cryo- and Plastic Sections for Immunoelectron Microscopy

    Cryo-Transmisson Electron Microscopy of Thin Vitrified Sections

    Preparation Methods for Quantitative X-Ray Microanalysis of intracellular Elements in Ultrathin Sections for Transmission Electron Microscopy: The Freeze-Dry, Resin-Embed Route

    High-Resolution Scanning Electron Microscopy in Cell Biology

    Part 5: Microdissection Techniques

    Microdissection-Based Techniques for the Determination of Cell Proliferation in Gastrointestinal Epithelium: Application to Animal and Human Studies

    Micromanipulation of Chromosomes Using Laser Microsurgery (Optical Scissors) and Laser-Induced Optical Forces [Optical Tweezers)

    Microdissection of Chromosomes and Microcloning

    Part 6: Histochemistry

    Mayer's Hematoxylin-Eosin: An Example of a Common Histological Staining Method

    Selected Enzyme Staining in Histochemistry

    Part 7: Antibodies

    Section A: Production of Antibodies

    Production of Polyclonal Antibodies in Rabbits

    Production of Mouse Monoclonal Antibodies

    Production of Human Monoclonal Antibodies via Fusion of Epstein-Barr Virus-Transformed Lymphocytes with Heteromyeloma

    Rapid Production of Antibodies in Chicken and Isolation from Eggs

    Section B: Purification and Labeling of Immunoglobulins

    Purification of Immunoglobulins

    Conjugation of Fluorescent Dyes to Antibodies

    Section C: Antibody Specificity

    Determination of Antibody Specificity by Western Blotting and Immunoprecipitation

    Western Blotting and Ligand Blotting Using Enhanced Chemiluminescence and Radioiodine Detection

    Enzyme-Linked Immunosorbent Assay

    A Simple Solid-Phase Mutual Inhibition Assay Using Biotinylated Antigen for Analyzing the Epitope Specificities of Monoclonal Antibodies

    DNA Immunoprecipitation: Application to Characterization of Target Sequences for a Human Centromere DNA-binding Protein (CENP-B)

    Part 8: Immunocytochemistry And Vital Staining of Cells

    Immunofluorescence Microscopy of Cultured Cells

    Immunofluorescence Microscopy of the Cytoskeleton: Double and Triple Immunofluorescence

    Immunocytochemistry of Frozen and of Paraffin Tissue Sections

    Fluorescent Labeling of Nascent RNA in the Cell Nucleus Using 5-Bromouridine 5'-Triphosphate

    Labeling of Endocytic Vesicles Using Fluorescent Probes for Fluid-Phase Endocytosis

    Labeling of the Endoplasmic Reticulum with DiO6 (3)

    Use of Fluorescent Analogs of Ceramide to Study the Golgi Apparatus of Animal Cells

    Vital Staining of Mitochondria with Rhodamine 123 and of Acidic Organelles with Acridine Orange

    Part 9: Intracellular Measurements

    Measuring Membrane Potential in Single Cells with Confocal Microscopy

    Measurement of Cytosolic pH in Single Cells by Dual-Excitation Fluorescence Spectrometry: Simultaneous Visualization Using Hoffman Modulation Contrast Optics

    Part 10: Cytogenetics and in Situ Hybridization

    Basic Cytogenetic Techniques: Culturing, Slide Making, and G-Banding

    Production of Viable Hybrids Between Adherent Cells

    Microcell-Mediated Chromosome Transfer: Selective Transfer and Retention of Single Human Chromosomes into Recipient Cells of Choice

    Microcell Transfer of Chromosomes from Mitotic Cells

    Chromosome Painting Using Degenerate Oligonucleotide-Primed Polymerase Chain Reaction-Amplified, Flow-Sorted Human Chromosomes

    Fluorescence in Situ Hybridization of Human and Mouse DNA Probes to Determine the Chromosomal Contents of Cell Lines and Tumors

    in Situ Hybridization Applicable to Abundantly Expressed mRNA Species

    In Situ Hybridization of Frozen Sections Using 3 5S-Riboprobes

    In Situ Detection of Human Papillomavirus DNA After Polymerase Chain Reaction Amplification

    Accurate Quantitation of mRNA Species by Polymerase Chain Reaction and Solid-Phase Minisequencing

Product details

  • No. of pages: 566
  • Language: English
  • Copyright: © Academic Press 1994
  • Published: January 1, 1994
  • Imprint: Academic Press
  • eBook ISBN: 9781483282459

About the Editor

Julio E Celis

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