Introduction
Unit 1.1 Basic Techniques for Mammalian Cell Tissue Culture
Unit 1.2 Media for Culture of Mammalian Cells
Unit 1.3 Aseptic Technique for Cell Culture
Unit 1.4 Sterilization and Filtration
Unit 1.5 Assessing and Controlling Microbial Contamination in Cell Cultures
Unit 1.6 Media and Culture of Yeast
Unit 1.7 BY-2 Cells: Culture and Transformation for Live Cell Imaging
Chapter 2 Preparation and Isolation of Cells
Introduction
Unit 2.1 Establishment of Fibroblast Cultures
Unit 2.2 Preparation and Culture of Human Lymphocytes
Unit 2.3 Preparation of Endothelial Cells
Unit 2.4 Generation of Continuously Growing B Cell Lines by Epstein-Barr Virus Transformation
Unit 2.5 Laser Capture Microdissection
Unit 2.6 Preparation of Human Epidermal Keratinocyte Cultures
Chapter 3 Subcellular Fractionation and Isolation of Organelles
Introduction
Unit 3.1 Overview of Cell Fractionation
Unit 3.2 Isolation of Rat Hepatocyte Plasma Membrane Sheets and Plasma Membrane Domains
Unit 3.3 Isolation of Mitochondria from Tissues and Cells by Differential Centrifugation
Unit 3.4 Purification of a Crude Mitochondrial Fraction by Density-Gradient Centrifugation
Unit 3.5 Isolation of Peroxisomes from Tissues and Cells by Differential and Density Gradient
Centrifugation
Unit 3.6 Isolation of Lysosomes from Tissues and Cells by Differential and Density Gradient
Centrifugation
Unit 3.7 Overview of Subcellular Fractionation Procedures for the Yeast Saccharomyces cerevisiae
Unit 3.8 Isolation of Subcellular Fractions from the Yeast Saccharomyces cerevisiae
Unit 3.9 Isolation of Golgi Membranes from Tissues and Cells by Differential and Density Gradient
Centrifugation
Unit 3.10 Isolation of Nuclei and Nuclear Membranes From Animal Tissues
Unit 3.11 Free-Flow Electrophoretic Analysis of Endosome Subpopulations of Rat Hepatocytes
Chapter 4 Microscopy
Introduction
Unit 4.1 Proper Alignment and Adjustment of the Light Microscope
Unit 4.2 Fluorescence Microscopy
Unit 4.3 Immunofluorescence Staining
Unit 4.4 Fluorescent Staining of Subcellular Organelles: ER, Golgi Complex, and Mitochondria
Unit 4.5 Basic Confocal Microscopy
Unit 4.6 Immunoperoxidase Methods for Localization of Antigens in Cultured Cells and Tissues
Unit 4.7 Cryo-Immunogold Electron Microscopy
Unit 4.8 Correlative Video Light/Electron Microscopy
Unit 4.9 Polarization Microscopy
Unit 4.10 Fluorescent Speckle Microscopy (FSM) of Microtubules and Actin in Living Cells
Unit 4.11 Two-Photon Excitation Microscopy for the Study of Living Cells and Tissues
Unit 4.12 Total Internal Reflection Fluorescence Microscopy for High-Resolution Imaging of Cell-Surface
Events
Unit 4.13 Fluorescent Labeling of Yeast
Unit 4A Organelle Atlas: Appendix to Chapter 4
Chapter 5 Characterization of Cellular Proteins
Introduction
Unit 5.1 Overview of the Physical State of Proteins Within Cells
Unit 5.2 Determining the Topology of an Integral Membrane Protein
Unit 5.3 Determination of Molecular )
Unit 5.4 Analysis of the Association of Proteins with Membranes
Unit 5.5 Determination of Molecular )
Unit 5.6 Identification of Proteins in Complex Mixtures Using Liquid Chromatography and Mass
Spectrometry
Chapter 6 Electrophoresis and Immunoblotting
Introduction
Unit 6.1 One-Dimensional SDS Gel Electrophoresis of Proteins
Unit 6.2 Immunoblotting and Immunodetection
Unit 6.3 Detection and Quantitation of Radiolabeled Proteins in Gels and Blots
Unit 6.4 Two-Dimensional Gel Electrophoresis
Unit 6.5 One-Dimensional Electrophoresis Using Nondenaturing Conditions
Unit 6.6 Staining Proteins in Gels
Unit 6.7 Agarose Gel Electrophoresis of Proteins
Unit 6.8 Fluorescence Detection of Glycoproteins in Gels and on Electroblots
Unit 6.9 Digital Electrophoresis Analysis
Chapter 7 Protein Labeling and Immunoprecipitation
Introduction
Unit 7.1 Metabolic Labeling with Amino Acids
Unit 7.2 Immunoprecipitation
Unit 7.3 Metabolic Labeling with Sulfate
Unit 7.4 Metabolic Labeling with Fatty Acids
Unit 7.5 Metabolic Labeling of Prenyl and Carboxyl-Methyl Groups
Unit 7.6 Metabolic Labeling and Immunoprecipitation of Yeast Proteins
Unit 7.7 Metabolic Labeling and Immunoprecipitation of Drosophila Proteins
Unit 7.8 Metabolic Labeling of Glycoproteins with Radioactive Sugars
Unit 7.9 Analysis of Oxidative Modification of Proteins
Unit 7.10 Radioiodination of Cellular Proteins
Chapter 8 Cell Cycle Analysis
Introduction
Unit 8.1 Overview of the Cell Cycle
Unit 8.2 Assays for CDK Activity and DNA Replication in the Cell Cycle
Unit 8.3 Methods for Synchronizing Cells at Specific Stages of the Cell Cycle
Unit 8.4 Determining Cell Cycle Stages by Flow Cytometry
Unit 8.5 Centrifugal Elutriation to Obtain Synchronous Populations of Cells
Chapter 9 Cell Adhesion
Introduction
Unit 9.1 Cell-Substrate Adhesion Assays
Unit 9.2 Quantitative Measurement of Cell Adhesion Using Centrifugal Force
Unit 9.3 Cadherin-Dependent Cell-Cell Adhesion
Unit 9.4 Analyzing Integrin-Dependent Adhesion
Unit 9.5 Analysis of Cell-Cell Contact Mediated by Ig Superfamily Cell Adhesion Molecules
Unit 9.6 Measurement of Adhesion Under Flow Conditions
Chapter 10 Extracellular Matrix
Introduction
Unit 10.1 Overview of Extracellular Matrix
Unit 10.2 Preparation of Basement Membrane Components from EHS Tumors
Unit 10.3 Preparation of Gelled Substrates
Unit 10.4 Preparation of Extracellular Matrices Produced by Cultured Corneal Endothelial and PF-HR9
Endodermal Cells
Unit 10.5 Purification of Fibronectin
Unit 10.6 Purification of Vitronectin
Unit 10.7 Proteoglycan Isolation and Analysis
Unit 10.8 Matrix Metalloproteinases
Unit 10.9 Preparation of Extracellular Matrices Produced by Cultured Fibroblasts
Unit 10.10 Purification and Analysis of Thrombospondin-1
Unit 10.11 Purification of SPARC/Osteonectin
Chapter 11 In Vitro Reconstitution
Introduction
Unit 11.1 Overview of Eukaryotic In Vitro Translation and Expression Systems
Unit 11.2 In Vitro Translation
Unit 11.3 In Vitro Analysis of Endoplasmic-Reticulum-to-Golgi Transport in Mammalian Cells
Unit 11.4 Cotranslational Translocation of Proteins into Canine Rough Microsomes
Unit 11.5 In Vitro Analysis of SV40 DNA Replication
Unit 11.6 In Vitro Transcription
Unit 11.7 Nuclear Import in Digitonin-Permeabilized Cells
Unit 11.8 In Vitro Translation Using HeLa Extract
Unit 11.9 Analysis of Eukaryotic Translation in Purified and Semipurified Systems
Unit 11.10 Preparation and Use of Interphase Xenopus Egg Extracts
Unit 11.11 Analysis of the Cell Cycle Using Xenopus Egg Extracts
Unit 11.12 Analysis of Apoptosis Using Xenopus Egg Extracts
Unit 11.13 Mitotic Spindle Assembly In Vitro
Unit 11.14 Analysis of RNA Export Using Xenopus Oocytes
Unit 11.15 In Vitro Analysis of Peroxisomal Protein Import
Unit 11.16 In Vitro Analysis of Chloroplast Protein Import
Unit 11.17 In Vitro RNA Splicing in Mammalian Cell Extracts
Chapter 12 Cell Motility
Introduction
Unit 12.1 Chemotaxis Assays for Eukaryotic Cells
Unit 12.2 Invasion Assays
Unit 12.3 Cell Traction
Unit 12.4 Cell Wound Assays
Unit 12.5 Dictyostelium Cell Dynamics
Unit 12.6 Optical Microscopy—Based Migration Assay for Human Neutrophils
Unit 12.7 Actin-Based Motility Assay
Chapter 13 Organelle Motility
Introduction
Unit 13.1 Microtubule/Organelle Motility Assays
Unit 13.2 In Vitro Motility Assay to Study Translocation of Actin by Myosin
Unit 13.3 Organelle Motility in Plant Cells: Imaging Golgi and ER Dynamics with GFP
Unit 13.4 Movement of Nuclei
Unit 13.5 Measuring Dynamics of Nuclear Proteins by Photobleaching
Chapter 14 Signal Transduction: Protein Phosphorylation
Introduction
Unit 14.1 Overview of Protein Phosphorylation
Unit 14.2 Immunological Detection of Phosphorylation
Unit 14.3 Detection of MAP Kinase Signaling
Unit 14.4 Labeling Cultured Cells with 32Pi and Preparing Cell Lysates for Immunoprecipitation
Unit 14.5 Phosphoamino Acid Analysis
Unit 14.6 Determination of Akt/PKB Signaling
Chapter 15 Protein Trafficking
Introduction
Unit 15.1 Overview of Protein Trafficking in the Secretory and Endocytic Pathways
Unit 15.2 Use of Glycosidases to Study Protein Trafficking
Unit 15.3 Endocytosis: Biochemical Analyses
Unit 15.4 Determining Protein Transport to the Plasma Membrane
Unit 15.5 Analysis of Membrane Traffic in Polarized Epithelial Cells
Unit 15.6 Analysis of Protein Folding and Oxidation in the Endoplasmic Reticulum
Unit 15.7 Measurements of Phagocytosis and Phagosomal Maturation
Chapter 16 Antibodies as Cell Biological Tools
Introduction
Unit 16.1 Production of Monoclonal Antibodies
Unit 16.2 Production of Polyclonal Antisera
Unit 16.3 Purification of Immunoglobulin G
Unit 16.4 Fragmentation of Immunoglobulin G
Unit 16.5 Antibody Conjugates for Cell Biology
Unit 16.6 Production of Antibodies That Recognize Specific Tyrosine-Phosphorylated Peptides
Chapter 17 Macromolecular Interactions in Cells
Introduction
Unit 17.1 Imaging Protein-Protein Interactions by Fluorescence Resonance Energy Transfer (FRET)
Microscopy
Unit 17.2 Identification of Protein Interactions by Far Western Analysis
Unit 17.3 Interaction Trap/Two-Hybrid System to Identify Interacting Proteins
Unit 17.4 Mapping Protein-Protein Interactions with Phage-Displayed Combinatorial Peptide Libraries
Unit 17.5 Protein-Protein Interactions Identified by Pull-Down Experiments and Mass Spectrometry
Unit 17.6 Measuring Protein Interactions by Optical Biosensors
Unit 17.7 Chromatin Immunoprecipitation for Determining the Association of Proteins with Specific
Genomic Sequences In Vivo
Unit 17.8 Isothermal Titration Calorimetry
Chapter 18 Cellular Aging and Death
Introduction
Unit 18.1 Current Concepts in Cell Death
Unit 18.2 Analysis of Caspase Activation During Apoptosis
Unit 18.3 Assessment of Apoptosis and Necrosis by DNA Fragmentation and Morphological Criteria
Unit 18.4 Quantitative Fluorescence In Situ Hybridization (Q-FISH)
Unit 18.5 Analysis of Mitochondrial Dysfunction During Cell Death
Unit 18.6 Analysis of Telomeres and Telomerase
Unit 18.7 Nonisotopic Methods for Determination of Poly(ADP-Ribose) Levels and Detection of Poly(ADPRibose)
Polymerase
Unit 18.8 Flow Cytometry of Apoptosis
Chapter 19 Whole Organism and Tissue Analysis
Introduction
Unit 19.1 Overview of Metastasis Assays
Unit 19.2 Tail Vein Assay of Cancer Metastasis
Unit 19.3 Microanalysis of Gene Expression in Tissues Using T7-SAGE: Serial Analysis of Gene Expression
After High-Fidelity T7-Based RNA Amplification
Unit 19.4 SAGE Analysis from 1 μg of Total RNA
Unit 19.5 The Chick Chorioallantoic Membrane as an In Vivo Angiogenesis Model
Unit 19.6 Experimental Metastasis Assays in the Chick Embryo
Chapter 20 Expression and Introduction of Macromolecules into Cells
Introduction
Unit 20.1 Direct Introduction of Molecules into Cells
Unit 20.2 Protein Transduction: Generation of Full-Length Transducible Proteins Using the TAT
System
Unit 20.3 Calcium Phosphate Transfection
Unit 20.4 Transfection Using DEAE-Dextran
Unit 20.5 Transfection by Electroporation
Unit 20.6 Transfection of Cultured Eukaryotic Cells Using Cationic Lipid Reagents
Unit 20.7 Optimization of Transfection
Chapter 21 Fluorescent Protein Technology
Introduction
Unit 21.1 Measuring Protein Mobility by Photobleaching GFP Chimeras in Living Cells
Unit 21.2 Fluorescence Localization After Photobleaching (FLAP)
Chapter 22 Cell Biology of Chromosomes and Nuclei
Introduction
Unit 22.1 Overview of Cytogenetic Chromosome Analysis
Unit 22.2 Preparation of Cytogenetic Specimens from Tissue Samples
Unit 22.3 Traditional Banding of Chromosomes for Cytogenetic Analysis
Unit 22.4 Fluorescence In Situ Hybridization (FISH)
Unit 22.5 Multi-Color FISH Techniques
Appendix 1 Useful Information and Data
1A Useful Measurements and Data
1B Compendium of Drugs Commonly Used in Cell Biology Research
1C Identification of Motifs in Protein Sequences
1D Safe Use of Radioisotopes
1E Absorption and Emission Maxima for Common Fluorophores
1F Importing Biological Materials
1G Centrifuges and Rotors
1H Internet Basics for Biologists
Appendix 2 Laboratory Stock Solutions and Equipment
2A Common Stock Solutions, Buffers, and Media
2B Medium Formulations
2C Standard Laboratory Equipment
Appendix 3 Commonly Used Techniques
3A Molecular Biology Techniques
3B Spectrophotometric Determination of Protein Concentration
3C Dialysis and Concentration of Protein Solutions
3D Quantification of DNA and RNA with Absorption and Fluorescence Spectroscopy
3E Silanizing Glassware
3F Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization
3G Micro RT-PCR
3H The Colorimetric Detection and Quantitation of Total Protein
SUPPLIERS APPENDIX
Selected Suppliers of Reagents and Equipment
