© 1998 • 2136 pp., illus., color plates, appendices, index
Plastic comb binding (three-book set) • ISBN  0-87969-521-8

This title also available in: Cloth (three-book set)

Having identified a gene product, how do you determine what it does? The answer lies in Cells, a new manual designed to do for studies of cell biology what Cold Spring Harbor's Molecular Cloning has done for molecular biology.

• Sets the standard for techniques of proven bench reliability needed by all biomedical scientists studying cellular structure and function.
• Delivers consistent, precisely crafted step-by-step protocols in an accessible format, with essential background details and in-depth advice on pitfalls and problem solving.
• Created by three distinguished cell biologist/educators, from the contributions of over 180 leading cell biologists.
• Complete with more than 300 expertly selected and superbly reproduced illustrations, over 70 in color.

Volume 1: Culture and Biochemical Analysis of Cells
Volume 2: Light Microscopy and Cell Structure
Volume 3: Subcellular Localization of Genes and Their Products

  "Cells … an indispensable addition to any modern life science laboratory."
      —GÜNTER BLOBEL, Rockefeller University

  "This is another bible for the modern era of biological researchers, joining the two crucial staples Molecular Cloning and Antibodies…. This three volume text fills a gap that has cried for attention, to produce an authoritative and straightforward text that describes the theory and practice of the principal basic-and-beyond techniques for molecular cell biology."
      —Trends in Genetics

  "This manual is a ‘must’ for any laboratory planning to use cell biology techniques. It will be of great use both to beginners and for those who have been chained to the bench a little longer! … so interesting and well written that I and members of the laboratory found ourselves reading sections for methods that we might never consider…."
      —Trends in Cell Biology

Volume I. Culture and Biochemical Analysis of Cells

Section 1: Cell Culture and Analysis

• Mammalian Cell Culture
• Growth and Manipulation of Cells in Culture
• Extracellular Matrix Components as Substrate in Cell and Tissue Culture
• Isolation and Culture of Fibroblasts
• Culture of Epithelial Cells
• Isolation of Human Capillary Endothelial Cells
• Isolation and Transformation of Lymphocytes
• Culture and In Vitro Differentiation of Mouse Embryonic Stem Cells
• Isolation and Culture of Primary Neural Cells
• Isolation and Culture of Chick Embryo Myoblasts
• Isolation and Culture of Neonatal Rat Cardiomyocytes
• Isolation and Culture of Adult Rat Ventricular Myocytes
• Fusion of Somatic Cells
• Cell Synchronization
• Apoptosis Assays
• Flow Cytometry
• Culture and Manipulation of Hypotrichous Ciliated Protozoa
• Culture and Manipulation of Tetrahymena
• Culture and Genetic Analysis of Chlamydomonas
• Culture and Analysis of Dictyostelium discoideum
• Growth and Transformation of Saccharomyces cerevisiae
• Growth and Transformation of Schizosaccharomyces pombe
• Culture of Marine Invertebrate Eggs

Section 2: Metabolic Labeling and Protein Modification

• Steady-State Labeling of HeLa Cells with ³²P Phosphate
• In Vitro Nuclear Run-on Analysis of mRNA Transcription
• Metabolic Radiolabeling of Proteins
• Metabolic Labeling of Cells with ³²P for Studies of Protein Phosphorylation
• Study of Protein Phosphorylation Using Permeabilized Cells and Organelles
• Protein Kinase Assays Using Exogenous Substrates
• Kinetics for Kinases: Simple Enzyme Kinetics Applied to Two-substrate Enzymes
• Simple Preparations of Protein Phosphatases
• Methylation and Prenylation of Proteins
• Protein Glycosylation

Section 3: Subcellular Fractionation

• Subcellular Fractionation
• Plasma Membrane Isolation Using the Cationic Colloidal Silica Isolation Technique
• Isolation of Desmosomes
• Subcellular Fractionation of Rough Microsomes
• Purification of Ribosomes, Ribosomal Subunits, and Polysomes
• Golgi Apparatus Isolation and Characterization
• Isolation of Peroxisomes (Microbodies) from Mammalian Tissues
• Isolation of Mitochondria from Cells and Tissues
• Isolation of Intact Chloroplasts
• Preparation of Nuclei from Tissue and Suspension Cultures
• The Nuclear Matrix: Preparation for Microscopy and Biochemical Analysis
• Permeabilized Cell Assay for Nuclear Protein Import
• Analysis of Nucleocytoplasmic Shuttling Using Transient Interspecies Heterokaryons
• Preparation and Characterization of the Nuclear Lamina and Nuclear Lamina-enriched Fractions
• Isolation of Nucleoli
• Chromosome Isolation for Biochemical and Morphological Analysis
• Purification and Southern Blotting Analysis of DNA
• Purification and Northern Blot Analysis of Total RNA from Cells and Tissues
• Spread Preparation of Xenopus Germinal Vesicle Contents
• Purification of Myosin and Actin from Muscle and Nonmuscle Cells
• Isolation of Microtubules, MAPs, and Microtubule-dependent Motor Proteins
• Isolation and Purification of Intermediate Filaments

Section 4: Protein Identification and Analysis

• Determination of Protein Concentration
• One-Dimensional Gel Electrophoresis of Proteins
• Two-Dimensional Gel Electrophoresis of Proteins
• Detection of Proteins by Staining
• Detection of Proteins by Autoradiography, Fluorography, and Phosphor Imaging
• One-Dimensional and Two-Dimensional Peptide Mapping
• Peptide Mapping and Peptide Purification by RP-HPLC
• Sequencing of Proteins and Peptides

Section 5: Protein Expression and Interactions

• Producing Proteins in a T7 Expression System
• Expression and Purification of Proteins Using GST Gene Fusion Vectors
• Baculovirus Expression: Generation of Recombinant Baculovirus DNA in E. coli Using a Baculovirus Shuttle Vector
• Mammalian Expression Vectors
• An Autoregulatory, Tetracycline-controlled System for Inducible Gene Expression in Mammalian Cells
• Two-hybrid System/Interaction Trap

Section 6: Antibodies as Tools in Cell Biology

• Overview of Antibody Purification and Labeling
• Epitope Tagging
• Immunoprecipitation
• Immunoblotting and Immunoblot Affinity Purification
• Human Autoantibodies and Characterization of Their Target Antigens

Volume II. Light Microscopy and Cell Structure

Section 7: Observation of Live Cells and Cellular Dynamics

• Observation of Live Cells in the Light Microscope
• Monitoring Molecular Dynamics In Vivo Using Fluorescence Techniques
• Phagokinetic Track Assay of Cell Locomotion in Tissue Culture
• Heterologous Expression of the Green Fluorescent Protein
• Fluorescence Photobleaching Techniques
• Imaging and Measurement of Intracellular Free Calcium in Living Cells
• Construction of Optical Tweezers
  [Selected Chapter online]

Section 8: Preparation of Macromolecules and Introduction into Cells

• Fluorescence Labeling of Antibodies and DNA Probes
• Quantitative Microinjection of Living Cells
• Injection of Xenopus Oocytes and Embryos
• Direct Injection of DNA into Rodent Muscle
• Transfection of DNA into Mammalian Cells
• Delivery of DNA into Mammalian Cells Using Cationic Liposomes
• Electroporation and Electrofusion
• Delivery of Antisense Oligonucleotides
• Use and Application of Adenovirus Expression Vectors
• Replication-defective Herpesvirus Amplicon Vectors and Their Use for Gene Transfer
• Transduction of Genes Using Retroviral Vectors
• Lineage Analysis Using Retroviral Vectors

Section 9: Light and Epifluorescence Microscopy

• Principles of Light Microscopy
• Video Microscopy and Image Enhancement

Section 10: Confocal Microscopy, Multiphoton Microscopy, and Deconvolution

• Confocal Microscopy and Deconvolution Techniques
• Multiphoton Excitation Fluorescence Microscopy

Volume III. Subcellular Localization of Genes and Their Products

Section 11: Visualization of Organelles, Proteins, and Gene Expression

• Preparation of Cells and Tissues for Fluorescence Microscopy
• Detection of beta-Galactosidase and Alkaline Phosphatase Activities in Tissues
• Immunolocalization of Proteins Using Enzyme Detection
• Nonimmunological Fluorescent Labeling of Cellular Structures
• Introduction to Immunofluorescence Microscopy
• Immunostaining of Microtubules, Microtubule-associated Proteins, and Intermediate Filaments
• Immunofluorescence Localization of Actin
• Immunofluorescence Localization of Nuclear Proteins
• Immunofluorescence Methods for Saccharomyces cerevisiae
• Immunofluorescence Methods for Drosophila Tissues
• Immunofluorescence Methods for C. elegans
• Analyzing DNA Replication: Nonisotopic Labeling
• Analyzing RNA Synthesis: Nonisotopic Labeling

Section 12: In Situ Hybridization

• Fluorescence In Situ Hybridization to DNA
• Comparative Genomic Hybridization
• Chromosome Analysis by Spectral Karyotyping
• In Situ Hybridization of DNA and Nuclear RNA in Tissue Squashes Using ³H-labeled Probes
• Whole-Mount Fluorescence In Situ Hybridization to Drosophila Chromosomal DNA
• In Situ Hybridization to RNA
• Whole-Mount In Situ Detection of RNAs in Vertebrate Embryos and Isolated Organs
• Whole-Mount In Situ Detection of RNAs in Xenopus Embryos
• In Situ Polymerase Chain Reaction

Section 13: Electron Microscopy

• Image Production Using Transmission Electron Microscopy
• Preparative Methods for Transmission Electron Microscopy
• Image Production Using Scanning Electron Microscopy
• Preparative Methods for Scanning Electron Microscopy
• Scanning Transmission Electron Microscopy
• Electron Microscopy of DNA and DNA-binding Proteins
• Snapshop Blotting of Nucleic Acids and Nucleoprotein Complexes
• Cytochemical Staining and Enzyme Detection for Electron Microscopy
• Immunoelectron Microscopy
• Rapid Freezing of Cells and Molecules
• Freeze Substitution
• Immunocytochemistry on Ultrathin Cryosections
• Freeze Fracture and Freeze-Fracture Cytochemistry

Appendices
Index