Current Protocols in Cell Biology

Last Update: March 01, 2013

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Chapter 1 Cell Culture
- Introduction
- Unit 1.1 Basic Techniques in 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
- Unit 2.7 Preparation and Coculture of Neurons and Glial Cells
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
- Unit 3.12 Isolation of Synaptic Vesicles
- Unit 3.13 Isolation of Clathrin‐Coated Vesicles by Differential and Density Gradient Centrifugation
- Unit 3.14 Isolation of Melanosomes
- Unit 3.15 Isolation of Lipid Droplets from Cells by Density Gradient Centrifugation
- Unit 3.16 Isolation of Mast Cell Granules
- Unit 3.17 Immunoisolation of Centrosomes from Drosophila melanogaster
- Unit 3.18 Isolation of Zymogen Granules from Rat Pancreas
- Unit 3.19 Isolation of Glyoxysomes from Pumpkin Cotyledons
- Unit 3.20 Isolation of GLUT4 Storage Vesicles
- Unit 3.21 Isolation of Intestinal Brush‐Border Membranes
- Unit 3.22 Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological Fluids
- Unit 3.23 Isolation of Intermediate Filaments
- Unit 3.24 Isolation of T‐Tubules from Skeletal Muscle
- Unit 3.25 Isolation of Myelin
- Unit 3.26 Isolation of Renal Brush Borders
- Unit 3.27 Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria‐Associated Membrane Fractions from Transfected Cells and from Human Cytomegalovirus‐Infected Primary Fibroblasts
- Unit 3.28 Isolation of Amyloplasts
- Unit 3.29 Isolation of Microtubules and Microtubule Proteins
- Unit 3.30 Purification of Intact Chloroplasts from Arabidopsis and Spinach Leaves by Isopycnic Centrifugation
- Unit 3.31 Isolation of Neuromelanin Granules
- Unit 3.32 Isolation of Dense Core Secretory Vesicles from Pancreatic Endocrine Cells by Differential and Density Gradient Centrifugation
- Unit 3.33 Isolation and Biochemical Characterization of Amyloid Plaques and Paired Helical Filaments
- Unit 3.34 Isolation of Legionella‐Containing Vacuoles by Immuno‐Magnetic Separation
- Unit 3.35 Isolation of Platelet Granules
- Unit 3.36 Isolation of Nucleoli
- Unit 3.37 Isolation of Cytotoxic T Cell and NK Granules and Purification of Their Effector Proteins
- Unit 3.38 Isolation of Aggresomes and Other Large Aggregates
- Unit 3.39 Isolation of Chromaffin Granules
- Unit 3.40 Purification of Ribosomes from Human Cell Lines
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 4.14 Fluorescence Lifetime Imaging Microscopy
- Unit 4.15 Biological Second and Third Harmonic Generation Microscopy
- Unit 4.16 Analyzing Real‐Time Video Microscopy: The Dynamics and Geometry of Vesicles and Tubules in Endocytosis
- Unit 4.17 Scanning Electron Microscopy of Cell Surface Morphology
- Unit 4.18 Fluorescence Imaging Techniques for Studying Drosophila Embryo Development
- Unit 4.19 Quantitative Colocalization Analysis of Confocal Fluorescence Microscopy Images
- Unit 4.20 Visualizing Protease Activity in Living Cells: From Two Dimensions to Four Dimensions
- Unit 4.21 Photoactivated Localization Microscopy (PALM) of Adhesion Complexes
- Unit 4.22 Culturing MDCK Cells in Three Dimensions for Analyzing Intracellular Dynamics
- Unit 4.23 Interference Reflection Microscopy
- Unit 4.24 Fluorescence Correlation Spectroscopy in Living Cells: A Practical Approach
- Unit 4.25 Analysis of Mitochondrial Dynamics and Functions Using Imaging Approaches
- Unit 4.26 Visualization of Live Primary Cilia Dynamics Using Fluorescence Microscopy
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 Size by Zonal Sedimentation Analysis on Sucrose Density Gradients
- Unit 5.4 Analysis of the Association of Proteins with Membranes
- Unit 5.5 Determination of Molecular Size by Size‐Exclusion Chromatography (Gel Filtration)
- Unit 5.6 Identification of Proteins in Complex Mixtures Using Liquid Chromatography and Mass Spectrometry
- Unit 5.7 Determining Membrane Protein Topologies in Single Cells and High‐Throughput Screening Applications
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
- Unit 6.10 Two‐Dimensional Blue Native Polyacrylamide Gel Electrophoresis
- Unit 6.11 Measurement of Oxidatively‐Induced Clustered DNA Lesions Using a Novel Adaptation of Single Cell Gel Electrophoresis (Comet Assay)
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
- Unit 7.11 Metabolic Labeling with Noncanonical Amino Acids and Visualization by Chemoselective Fluorescent Tagging
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
- Unit 8.6 Dynamic Proliferation Assessment in Flow Cytometry
- Unit 8.7 Cell Biological Analysis of DT40 Knockout Cell Lines for Cell‐Cycle Genes
- Unit 8.8 Analysis of Protein Turnover by Quantitative SNAP‐Based Pulse‐Chase Imaging
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 and Primary Fibroblasts
- Unit 10.10 Purification and Analysis of Thrombospondin‐1
- Unit 10.11 Purification of SPARC/Osteonectin
- Unit 10.12 Analysis of Fibronectin Matrix Assembly
- Unit 10.13 Non‐Radioactive Quantification of Fibronectin Matrix Assembly
- Unit 10.14 Use of Hyaluronan‐Derived Hydrogels for Three‐Dimensional Cell Culture and Tumor Xenografts
- Unit 10.15 Generation of Micropatterned Substrates Using Micro Photopatterning
- Unit 10.16 Preparation of Hydrogel Substrates with Tunable Mechanical Properties
- Unit 10.17 Engineering Three‐Dimensional Collagen Matrices to Provide Contact Guidance during 3D Cell Migration
- Unit 10.18 Imaging Cells in Three‐Dimensional Collagen Matrix
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
- Unit 11.18 Endocytosis Assays in Intact and Permeabilized Cells
- Unit 11.19 In Vitro Analysis of Yeast Mitochondrial Protein Import
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
- Unit 12.8 In Vivo Marking of Single Cells in Chick Embryos Using Photoactivation of GFP
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
- Unit 13.6 Functional Characterization of Proteins Regulating Actin Assembly
Chapter 14 Signal Transduction: Protein Phosphorylation
- Introduction
- Unit 14.1 Overview of Protein Phosphorylation
- Unit 14.2 Immunological Detection of Phosphorylation
- Unit 14.3 The Detection of MAPK 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
- Unit 14.7 Analyzing FAK and Pyk2 in Early Integrin Signaling Events
- Unit 14.8 Rho GTPase Activation Assays
- Unit 14.9 In Vitro GEF and GAP Assays
- Unit 14.10 In Vivo Imaging of Signal Transduction Cascades with Probes Based on Förster Resonance Energy Transfer (FRET)
- Unit 14.11 Biosensors for Characterizing the Dynamics of Rho Family GTPases in Living Cells
- Unit 14.12 Analysis of Arf GTP‐Binding Protein Function in Cells
- Unit 14.13 Allosteric Activation of Kinases: Design and Application of RapR Kinases
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
- Unit 15.8 Analysis of Protein Transport to Lysosomes
- Unit 15.9 Studies of the Ubiquitin Proteasome System
- Unit 15.10 Measuring Retrograde Transport to the Trans‐Golgi Network
- Unit 15.11 Assays for Regulated Exocytosis of Mast Cell Granules
- Unit 15.12 Analysis of Regulated Secretion Using PC12 Cells
- Unit 15.13 Analysis of Endocytic Trafficking by Single‐Cell Fluorescence Ratio Imaging
- Unit 15.14 Quantitative Analysis of Endocytosis and Turnover of Epidermal Growth Factor (EGF) and EGF Receptor
- Unit 15.15 Documenting GLUT4 Exocytosis and Endocytosis in Muscle Cell Monolayers
- Unit 15.16 Measurement of Autophagic Activity in Mammalian Cells
- Unit 15.17 Analysis of Polarized Membrane Traffic in Hepatocytes and Hepatic Cell Lines
- Unit 15.18 Analysis of Rab GTPases
- Unit 15.19 Synchronizing Protein Transport in the Secretory Pathway
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
- Unit 17.9 Rational Design and Evaluation of FRET Experiments to Measure Protein Proximities in Cells
- Unit 17.10 Identification and Analysis of Multiprotein Complexes Through Chemical Crosslinking
- Unit 17.11 Visualization of RNA Using Fluorescence Complementation Triggered by Aptamer‐Protein Interactions (RFAP) in Live Bacterial Cells
- Unit 17.12 Use of In Vivo Biotinylation for Chromatin Immunoprecipitation
- Unit 17.13 Approaches to Studying Arf GAPs in Cells: In Vitro Assay with Isolated Focal Adhesions
- Unit 17.14 Chromatin Immunoprecipitation of Adult Murine Cardiomyocytes
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(ADP‐Ribose) Polymerase
- Unit 18.8 Flow Cytometry of Apoptosis
- Unit 18.9 Analysis of Cellular Senescence in Culture In Vivo: The Senescence‐Associated β‐Galactosidase Assay
- Unit 18.10 High‐Throughput Live Cell Imaging 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
- Unit 19.7 Imaging Tumor Cell Movement In Vivo
- Unit 19.8 Embryonic Organ Culture
- Unit 19.9 Three‐Dimensional Tissue Models of Normal and Diseased Skin
- Unit 19.10 Overview: Engineering Transgenic Constructs and Mice
- Unit 19.11 Generation of Transgenic Mice
- Unit 19.12 Overview: Generation of Gene Knockout Mice
- Unit 19.13 Manipulation of Mouse Embryonic Stem Cells for Knockout Mouse Production
- Unit 19.14 Generation of Gene Knockout Mice by ES Cell Microinjection
- Unit 19.15 Chick Embryo Culture and Electroporation
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
- Unit 20.8 Inducible Gene Expression Using an Autoregulatory, Tetracycline‐Controlled System
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)
- Unit 21.3 Visualization of Protein Interactions in Living Cells Using Bimolecular Fluorescence Complementation (BiFC) Analysis
- Unit 21.4 Design and Use of Fluorescent Fusion Proteins in Cell Biology
- Unit 21.5 The Fluorescent Protein Color Palette
- Unit 21.6 Photoactivation and Imaging of Photoactivatable Fluorescent Proteins
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
- Unit 22.6 Comparative Genomic Hybridization
- Unit 22.7 Sister Chromatid Exchange
- Unit 22.8 Detection of Mitotic Figures and Components of the Mitotic Machinery
- Unit 22.9 Assembly and Micromanipulation of Xenopus In Vitro–Assembled Mitotic Chromosomes
- Unit 22.10 Replication Labeling with Halogenated Thymidine Analogs
- Unit 22.11 Assays for Ribosomal RNA Processing and Ribosome Assembly
- Unit 22.12 Visualization and Measurement of DNA Methyltransferase Activity in Living Cells
- Unit 22.13 Monitoring mRNA Export
- Unit 22.14 Analysis of DNA Replication in Saccharomyces cerevisiae by Two‐Dimensional and Pulsed‐Field Gel Electrophoresis
- Unit 22.15 Analysis of Interactions Between Genomic Loci Through Chromosome Conformation Capture (3C)
- Unit 22.16 Assays to Measure Nuclear Mechanics in Interphase Cells
Chapter 23 Stem Cells
- Introduction
- Unit 23.1 Stem Cells: An Overview
- Unit 23.2 Mouse Embryonic Stem Cell Derivation, and Mouse and Human Embryonic Stem Cell Culture and Differentiation as Embryoid Bodies
- Unit 23.3 Maintenance and In Vitro Differentiation of Mouse Embryonic Stem Cells to Form Blood Vessels
- Unit 23.4 Differentiation of Mouse Embryonic Stem Cells and of Human Adult Stem Cells into Adipocytes
- Unit 23.5 Induction of ES Cell–Derived Cartilage Formation
- Unit 23.6 Hematoendothelial Differentiation of Human Embryonic Stem Cells
- Unit 23.7 Neural Differentiation of Human ES Cells
- Unit 23.8 Cardiac Differentiation of Human Embryonic Stem Cells and their Assembly into Engineered Heart Muscle
Chapter 24 Lipids
- Introduction
- Unit 24.1 Using Fluorescent Sphingolipid Analogs to Study Intracellular Lipid Trafficking
- Unit 24.2 Fluorescent Detection of Lipid Droplets and Associated Proteins
- Unit 24.3 Making Giant Unilamellar Vesicles via Hydration of a Lipid Film
- Unit 24.4 Visualization of Cellular Phosphoinositide Pools with GFP‐Fused Protein‐Domains
Chapter 25 Nanotechnology
- Introduction
- Unit 25.1 In Vivo Imaging Using Quantum Dot–Conjugated Probes
- Unit 25.2 Fabrication and Application of Nanofibrous Scaffolds in Tissue Engineering
Chapter 26 Viruses
- Introduction
- Unit 26.1 Production of Papillomavirus‐Based Gene Transfer Vectors
- Unit 26.2 BK Virus (BKV): Infection, Propagation, Quantitation, Purification, Labeling, and Analysis of Cell Entry
- Unit 26.3 Methods Used to Study Respiratory Virus Infection
- Unit 26.4 Compartmented Neuron Cultures for Directional Infection by Alpha Herpesviruses
- Unit 26.5 HIV‐1 Interactions with Cells: From Viral Binding to Cell‐Cell Transmission
- Unit 26.6 Methods for Monitoring Dynamics of Pulmonary RSV Replication by Viral Culture and by Real‐Time Reverse Transcription–PCR In Vivo: Detection of Abortive Viral Replication
- Unit 26.7 COS‐1 Cells as Packaging Host for Production of Lentiviruses
- Unit 26.8 Production of Lentiviral Vectors in Protein‐free Media
- Unit 26.9 Monitoring Viral‐Mediated Membrane Fusion Using Fluorescent Reporter Methods
- Unit 26.10 Evaluation of Antiseptic Antiviral Activity of Chemical Agents
- Unit 26.11 Rapid Titration of Viruses by Flow Cytometry
- Unit 26.12 An Enzymatic Assay for Detection of Viral Entry
- Unit 26.13 A Dual‐Chamber Model of the Female Genital Tract to Evaluate Epithelial Toxicity of Candidate Anti‐HIV Microbicides
- Unit 26.14 Methods for Growing and Titrating African Swine Fever Virus: Field and Laboratory Samples
Chapter 27 RNA‐Based Methods in Cell Biology
- Introduction
- Unit 27.1 Silencing of Gene Expression in Cultured Cells Using Small Interfering RNAs
- Unit 27.2 Gene Down‐Regulation with Short Hairpin RNAs and Validation of Specificity by Inducible Rescue in Mammalian Cells
- Unit 27.3 Analysis of Nonsense‐Mediated mRNA Decay in Saccharomyces cerevisiae
- Unit 27.4 Analysis of Nonsense‐Mediated mRNA Decay in Mammalian Cells
Appendix 1 Useful Information and Data
- Appendix 1A Useful Measurements and Data
- Appendix 1B Compendium of Drugs Commonly Used in Cell Biology Research
- Appendix 1C Identification of Motifs in Protein Sequences
- Appendix 1D Safe Use of Radioisotopes
- Appendix 1E Absorption and Emission Maxima for Common Fluorophores
- Appendix 1F Importing Biological Materials
- Appendix 1G Centrifuges and Rotors
- Appendix 1H Internet Basics for Biologists
Appendix 2 Laboratory Stock Solutions and Equipment
- Appendix 2A Common Stock Solutions, Buffers, and Media
- Appendix 2B Medium Formulations
- Appendix 2C Standard Laboratory Equipment
Appendix 3 Commonly Used Techniques
- Appendix 3A Molecular Biology Techniques
- Appendix 3B Spectrophotometric Determination of Protein Concentration
- Appendix 3C Dialysis and Concentration of Protein Solutions
- Appendix 3D Quantification of DNA and RNA with Absorption and Fluorescence Spectroscopy
- Appendix 3E Silanizing Glassware
- Appendix 3F Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization
- Appendix 3G Micro RT‐PCR
- Appendix 3H The Colorimetric Detection and Quantitation of Total Protein

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