Current Protocols in Cell Biology

Current Protocols in Cell Biology

Online ISBN: 9780471143031

DOI: 10.1002/0471143030

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  1. Preface
  2. Foreword
  3. Selected Suppliers of Reagents and Equipment
  4. Chapter 1 Cell Culture
    1. Introduction
    2. UNIT 1.1 Basic Techniques in Mammalian Cell Tissue Culture
    3. UNIT 1.2 Media for Culture of Mammalian Cells
    4. UNIT 1.3 Aseptic Technique for Cell Culture
    5. UNIT 1.4 Sterilization and Filtration
    6. UNIT 1.5 Assessing and Controlling Microbial Contamination in Cell Cultures
    7. UNIT 1.6 Media and Culture of Yeast
    8. UNIT 1.7 BY-2 Cells: Culture and Transformation for Live Cell Imaging
    9. UNIT 1.8 Isolation, Culture, and Transfection of Melanocytes
    10. UNIT 1.9 Isolation and Culture of Spinal Cord Motor Neurons
  5. Chapter 2 Preparation and Isolation of Cells
    1. Introduction
    2. UNIT 2.1 Establishment of Fibroblast Cultures
    3. UNIT 2.2 Preparation and Culture of Human Lymphocytes
    4. UNIT 2.3 Preparation of Endothelial Cells
    5. UNIT 2.4 Generation of Continuously Growing B Cell Lines by Epstein-Barr Virus Transformation
    6. UNIT 2.5 Laser Capture Microdissection
    7. UNIT 2.6 Preparation of Human Epidermal Keratinocyte Cultures
    8. UNIT 2.7 Preparation and Coculture of Neurons and Glial Cells
    9. UNIT 2.8 Isolation, Culture, and Transient Transformation of Plant Protoplasts
    10. UNIT 2.9 Standardized Isolation and Culture of Murine Liver Sinusoidal Endothelial Cells
  6. Chapter 3 Subcellular Fractionation and Isolation of Organelles
    1. Introduction
    2. UNIT 3.1 Fractionation of Subcellular Organelles
    3. UNIT 3.2 Isolation of Rat Hepatocyte Plasma Membrane Sheets and Plasma Membrane Domains
    4. UNIT 3.3 Isolation of Mitochondria from Tissues and Cells by Differential Centrifugation
    5. UNIT 3.4 Purification of a Crude Mitochondrial Fraction by Density-Gradient Centrifugation
    6. UNIT 3.5 Isolation of Peroxisomes from Tissues and Cells by Differential and Density Gradient Centrifugation
    7. UNIT 3.6 Isolation of Lysosomes from Tissues and Cells by Differential and Density Gradient Centrifugation
    8. UNIT 3.7 Overview of Subcellular Fractionation Procedures for the Yeast Saccharomyces cerevisiae
    9. UNIT 3.8 Isolation of Subcellular Fractions from the Yeast Saccharomyces cerevisiae
    10. UNIT 3.9 Isolation of Golgi Membranes from Tissues and Cells by Differential and Density Gradient Centrifugation
    11. UNIT 3.10 Isolation of Nuclei and Nuclear Membranes From Animal Tissues
    12. UNIT 3.11 Free-Flow Electrophoretic Analysis of Endosome Subpopulations of Rat Hepatocytes
    13. UNIT 3.12 Isolation of Synaptic Vesicles
    14. UNIT 3.13 Isolation of Clathrin-Coated Vesicles by Differential and Density Gradient Centrifugation
    15. UNIT 3.14 Isolation of Melanosomes
    16. UNIT 3.15 Isolation of Lipid Droplets from Cells by Density Gradient Centrifugation
    17. UNIT 3.16 Isolation of Mast Cell Granules
    18. UNIT 3.17 Immunoisolation of Centrosomes from Drosophila melanogaster
    19. UNIT 3.18 Isolation of Zymogen Granules from Rat Pancreas
    20. UNIT 3.19 Isolation of Glyoxysomes from Pumpkin Cotyledons
    21. UNIT 3.20 Isolation of GLUT4 Storage Vesicles
    22. UNIT 3.21 Isolation of Intestinal Brush-Border Membranes
    23. UNIT 3.22 Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological Fluids
    24. UNIT 3.23 Isolation of Intermediate Filaments
    25. UNIT 3.24 Isolation of T-Tubules from Skeletal Muscle
    26. UNIT 3.25 Isolation of Myelin
    27. UNIT 3.26 Isolation of Renal Brush Borders
    28. UNIT 3.27 Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria-Associated Membrane and Detergent Resistant Membrane Fractions from Transfected Cells and from Human Cytomegalovirus-Infected Primary Fibroblasts
    29. UNIT 3.28 Isolation of Amyloplasts
    30. UNIT 3.29 Isolation of Microtubules and Microtubule Proteins
    31. UNIT 3.30 Purification of Intact Chloroplasts from Arabidopsis and Spinach Leaves by Isopycnic Centrifugation
    32. UNIT 3.31 Isolation of Neuromelanin Granules
    33. UNIT 3.32 Isolation of Dense Core Secretory Vesicles from Pancreatic Endocrine Cells by Differential and Density Gradient Centrifugation
    34. UNIT 3.33 Isolation and Biochemical Characterization of Amyloid Plaques and Paired Helical Filaments
    35. UNIT 3.34 Isolation of Legionella-Containing Vacuoles by Immuno-Magnetic Separation
    36. UNIT 3.35 Isolation of Platelet Granules
    37. UNIT 3.36 Isolation of Nucleoli
    38. UNIT 3.37 Isolation of Cytotoxic T Cell and NK Granules and Purification of Their Effector Proteins
    39. UNIT 3.38 Isolation of Aggresomes and Other Large Aggregates
    40. UNIT 3.39 Isolation of Chromaffin Granules
    41. UNIT 3.40 Purification of Ribosomes from Human Cell Lines
    42. UNIT 3.41 Isolation of Chlamydomonas Flagella
    43. UNIT 3.42 Isolation of Primary Cilia by Shear Force
    44. UNIT 3.43 Proteasomes: Isolation and Activity Assays
  7. Chapter 4 Microscopy
    1. Introduction
    2. UNIT 4.1 Proper Alignment and Adjustment of the Light Microscope
    3. UNIT 4.2 Fluorescence Microscopy
    4. UNIT 4.3 Immunofluorescence Staining
    5. UNIT 4.4 Fluorescent Staining of Subcellular Organelles: ER, Golgi Complex, and Mitochondria
    6. UNIT 4.5 Basic Confocal Microscopy
    7. UNIT 4.6 Immunoperoxidase Methods for Localization of Antigens in Cultured Cells and Tissues
    8. UNIT 4.7 Cryo-Immunogold Electron Microscopy
    9. UNIT 4.8 Correlative Video Light/Electron Microscopy
    10. UNIT 4.9 Polarization Microscopy
    11. UNIT 4.10 Fluorescent Speckle Microscopy (FSM) of Microtubules and Actin in Living Cells
    12. UNIT 4.11 Two-Photon Excitation Microscopy for the Study of Living Cells and Tissues
    13. UNIT 4.12 Total Internal Reflection Fluorescence Microscopy for High-Resolution Imaging of Cell-Surface Events
    14. UNIT 4.13 Fluorescent Labeling of Yeast
    15. UNIT 4.14 Fluorescence Lifetime Imaging Microscopy
    16. UNIT 4.15 Biological Second and Third Harmonic Generation Microscopy
    17. UNIT 4.16 Analyzing Real-Time Video Microscopy: The Dynamics and Geometry of Vesicles and Tubules in Endocytosis
    18. UNIT 4.17 Scanning Electron Microscopy of Cell Surface Morphology
    19. UNIT 4.18 Fluorescence Imaging Techniques for Studying Drosophila Embryo Development
    20. UNIT 4.19 Quantitative Colocalization Analysis of Fluorescence Microscopy Images
    21. UNIT 4.20 Visualizing Protease Activity in Living Cells: From Two Dimensions to Four Dimensions
    22. UNIT 4.21 Photoactivated Localization Microscopy (PALM) of Adhesion Complexes
    23. UNIT 4.22 Culturing MDCK Cells in Three Dimensions for Analyzing Intracellular Dynamics
    24. UNIT 4.23 Interference Reflection Microscopy
    25. UNIT 4.24 Fluorescence Correlation Spectroscopy in Living Cells: A Practical Approach
    26. UNIT 4.25 Analysis of Mitochondrial Dynamics and Functions Using Imaging Approaches
    27. UNIT 4.26 Visualization of Live Primary Cilia Dynamics Using Fluorescence Microscopy
    28. UNIT 4.27 Image Correlation Spectroscopy for Measurements of Particle Densities and Colocalization
    29. UNIT 4.28 Freeze-Fracture Immunocytochemistry: Fracture-Label and Label-Fracture for the Localization of Membrane Proteins
    30. UNIT 4.29 Polarized Fluorescence Microscopy to Study Cytoskeleton Assembly and Organization in Live Cells
    31. UNIT 4.30 A Convenient Technique to Fix Suspension Cells on a Coverslip for Microscopy
    32. You have free access to this content
      UNIT 4A Organelle Atlas: Appendix to Chapter 4
  8. Chapter 5 Characterization of Cellular Proteins
    1. Introduction
    2. UNIT 5.1 Overview of the Physical State of Proteins Within Cells
    3. UNIT 5.2 Determining the Topology of an Integral Membrane Protein
    4. UNIT 5.3 Determination of Molecular Size by Zonal Sedimentation Analysis on Sucrose Density Gradients
    5. UNIT 5.4 Analysis of the Association of Proteins with Membranes
    6. UNIT 5.5 Determination of Molecular Size by Size-Exclusion Chromatography (Gel Filtration)
    7. UNIT 5.6 Identification of Proteins in Complex Mixtures Using Liquid Chromatography and Mass Spectrometry
    8. UNIT 5.7 Determining Membrane Protein Topologies in Single Cells and High-Throughput Screening Applications
  9. Chapter 6 Electrophoresis and Immunoblotting
    1. Introduction
    2. UNIT 6.1 One-Dimensional SDS Gel Electrophoresis of Proteins
    3. UNIT 6.2 Immunoblotting and Immunodetection
    4. UNIT 6.3 Detection and Quantitation of Radiolabeled Proteins in Gels and Blots
    5. UNIT 6.4 Two-Dimensional Gel Electrophoresis
    6. UNIT 6.5 One-Dimensional Electrophoresis Using Nondenaturing Conditions
    7. UNIT 6.6 Staining Proteins in Gels
    8. UNIT 6.7 Agarose Gel Electrophoresis of Proteins
    9. UNIT 6.8 Fluorescence Detection of Glycoproteins in Gels and on Electroblots
    10. UNIT 6.9 Digital Electrophoresis Analysis
    11. UNIT 6.10 Two-Dimensional Blue Native Polyacrylamide Gel Electrophoresis
    12. UNIT 6.11 Measurement of Oxidatively-Induced Clustered DNA Lesions Using a Novel Adaptation of Single Cell Gel Electrophoresis (Comet Assay)
  10. Chapter 7 Protein Labeling and Immunoprecipitation
    1. Introduction
    2. UNIT 7.1 Metabolic Labeling with Amino Acids
    3. UNIT 7.2 Immunoprecipitation
    4. UNIT 7.3 Metabolic Labeling with Sulfate
    5. UNIT 7.4 Metabolic Labeling with Fatty Acids
    6. UNIT 7.5 Metabolic Labeling of Prenyl and Carboxyl-Methyl Groups
    7. UNIT 7.6 Metabolic Labeling and Immunoprecipitation of Yeast Proteins
    8. UNIT 7.7 Metabolic Labeling and Immunoprecipitation of Drosophila Proteins
    9. UNIT 7.8 Metabolic Labeling of Glycoproteins with Radioactive Sugars
    10. UNIT 7.9 Analysis of Oxidative Modification of Proteins
    11. UNIT 7.10 Radioiodination of Cellular Proteins
    12. UNIT 7.11 Metabolic Labeling with Noncanonical Amino Acids and Visualization by Chemoselective Fluorescent Tagging
  11. Chapter 8 Cell Cycle Analysis
    1. Introduction
    2. UNIT 8.1 Overview of the Cell Cycle
    3. UNIT 8.2 Assays for CDK Activity and DNA Replication in the Cell Cycle
    4. UNIT 8.3 Methods for Synchronizing Cells at Specific Stages of the Cell Cycle
    5. UNIT 8.4 Determining Cell Cycle Stages by Flow Cytometry
    6. UNIT 8.5 Centrifugal Elutriation to Obtain Synchronous Populations of Cells
    7. UNIT 8.6 Dynamic Proliferation Assessment in Flow Cytometry
    8. UNIT 8.7 Cell Biological Analysis of DT40 Knockout Cell Lines for Cell-Cycle Genes
    9. UNIT 8.8 Analysis of Protein Turnover by Quantitative SNAP-Based Pulse-Chase Imaging
  12. Chapter 9 Cell Adhesion
    1. Introduction
    2. UNIT 9.1 Cell-Substrate Adhesion Assays
    3. UNIT 9.2 Quantitative Measurement of Cell Adhesion Using Centrifugal Force
    4. UNIT 9.3 Cadherin-Dependent Cell-Cell Adhesion
    5. UNIT 9.4 Analyzing Integrin-Dependent Adhesion
    6. UNIT 9.5 Analysis of Cell-Cell Contact Mediated by Ig Superfamily Cell Adhesion Molecules
    7. UNIT 9.6 Measurement of Adhesion Under Flow Conditions
    8. UNIT 9.7 In Situ Detection of Integrin Ligands
    9. UNIT 9.8 Isolation of Integrin-Based Adhesion Complexes
  13. Chapter 10 Extracellular Matrix
    1. Introduction
    2. UNIT 10.1 Overview of Extracellular Matrix
    3. UNIT 10.2 Preparation of Basement Membrane Components from EHS Tumors
    4. UNIT 10.3 Preparation of Gelled Substrates
    5. UNIT 10.4 Preparation of Extracellular Matrices Produced by Cultured Corneal Endothelial and PF-HR9 Endodermal Cells
    6. UNIT 10.5 Purification of Fibronectin
    7. UNIT 10.6 Purification of Vitronectin
    8. UNIT 10.7 Proteoglycan Isolation and Analysis
    9. UNIT 10.8 Matrix Metalloproteinases
    10. UNIT 10.9 Preparation of Extracellular Matrices Produced by Cultured and Primary Fibroblasts
    11. UNIT 10.10 Purification and Analysis of Thrombospondin-1
    12. UNIT 10.11 Purification of SPARC/Osteonectin
    13. UNIT 10.12 Analysis of Fibronectin Matrix Assembly
    14. UNIT 10.13 Non-Radioactive Quantification of Fibronectin Matrix Assembly
    15. UNIT 10.14 Use of Hyaluronan-Derived Hydrogels for Three-Dimensional Cell Culture and Tumor Xenografts
    16. UNIT 10.15 Generation of Micropatterned Substrates Using Micro Photopatterning
    17. UNIT 10.16 Preparation of Hydrogel Substrates with Tunable Mechanical Properties
    18. UNIT 10.17 Engineering Three-Dimensional Collagen Matrices to Provide Contact Guidance during 3D Cell Migration
    19. UNIT 10.18 Imaging Cells in Three-Dimensional Collagen Matrix
    20. UNIT 10.19 Preparation of High-Density Fibrillar Collagen Matrices That Mimic Desmoplastic Tumor Stroma
  14. Chapter 11 In Vitro Reconstitution
    1. Introduction
    2. UNIT 11.1 Overview of Eukaryotic In Vitro Translation and Expression Systems
    3. UNIT 11.2 In Vitro Translation
    4. UNIT 11.3 In Vitro Analysis of Endoplasmic-Reticulum-to-Golgi Transport in Mammalian Cells
    5. UNIT 11.4 Cotranslational Translocation of Proteins into Canine Rough Microsomes
    6. UNIT 11.5 In Vitro Analysis of SV40 DNA Replication
    7. UNIT 11.6 In Vitro Transcription
    8. UNIT 11.7 Nuclear Import in Digitonin-Permeabilized Cells
    9. UNIT 11.8 In Vitro Translation Using HeLa Extract
    10. UNIT 11.9 Analysis of Eukaryotic Translation in Purified and Semipurified Systems
    11. UNIT 11.10 Preparation and Use of Interphase Xenopus Egg Extracts
    12. UNIT 11.11 Analysis of the Cell Cycle Using Xenopus Egg Extracts
    13. UNIT 11.12 Analysis of Apoptosis Using Xenopus Egg Extracts
    14. UNIT 11.13 Mitotic Spindle Assembly In Vitro
    15. UNIT 11.14 Analysis of RNA Export Using Xenopus Oocytes
    16. UNIT 11.15 In Vitro Analysis of Peroxisomal Protein Import
    17. UNIT 11.16 In Vitro Analysis of Chloroplast Protein Import
    18. UNIT 11.17 In Vitro RNA Splicing in Mammalian Cell Extracts
    19. UNIT 11.18 Endocytosis Assays in Intact and Permeabilized Cells
    20. UNIT 11.19 In Vitro Analysis of Yeast Mitochondrial Protein Import
    21. UNIT 11.20 In Vitro Assays of Lipidation of Mammalian Atg8 Homologs
    22. UNIT 11.21 In Vitro Analysis of the Very-Low Density Lipoprotein Export from the Trans-Golgi Network
  15. Chapter 12 Cell Motility
    1. Introduction
    2. UNIT 12.1 Chemotaxis Assays for Eukaryotic Cells
    3. UNIT 12.2 Invasion Assays
    4. UNIT 12.3 Cell Traction
    5. UNIT 12.4 Cell Wound Assays
    6. UNIT 12.5 Dictyostelium Cell Dynamics
    7. UNIT 12.6 Optical Microscopy–Based Migration Assay for Human Neutrophils
    8. UNIT 12.7 Actin-Based Motility Assay
    9. UNIT 12.8 In Vivo Marking of Single Cells in Chick Embryos Using Photoactivation of GFP
    10. UNIT 12.9 Direct Measurement of Intracellular Pressure
    11. UNIT 12.10 Identification and Characterization of Tunneling Nanotubes for Intercellular Trafficking
  16. Chapter 13 Organelle Motility
    1. Introduction
    2. UNIT 13.1 Microtubule/Organelle Motility Assays
    3. UNIT 13.2 In Vitro Motility Assay to Study Translocation of Actin by Myosin
    4. UNIT 13.3 Organelle Motility in Plant Cells: Imaging Golgi and ER Dynamics with GFP
    5. UNIT 13.4 Movement of Nuclei
    6. UNIT 13.5 Measuring Dynamics of Nuclear Proteins by Photobleaching
    7. UNIT 13.6 Functional Characterization of Proteins Regulating Actin Assembly
    8. UNIT 13.7 Adhesive Micropatterns to Study Intermediate Filament Function in Nuclear Positioning
    9. UNIT 13.8 A Novel Assay to Identify the Trafficking Proteins that Bind to Specific Vesicle Populations
  17. Chapter 14 Signal Transduction: Protein Phosphorylation
    1. Introduction
    2. UNIT 14.1 Overview of Protein Phosphorylation
    3. UNIT 14.2 Immunological Detection of Phosphorylation
    4. UNIT 14.3 The Detection of MAPK Signaling
    5. UNIT 14.4 Labeling Cultured Cells with 32Pi and Preparing Cell Lysates for Immunoprecipitation
    6. UNIT 14.5 Phosphoamino Acid Analysis
    7. UNIT 14.6 Determination of Akt/PKB Signaling
    8. UNIT 14.7 Analyzing FAK and Pyk2 in Early Integrin Signaling Events
    9. UNIT 14.8 Rho GTPase Activation Assays
    10. UNIT 14.9 In Vitro GEF and GAP Assays
    11. UNIT 14.10 In Vivo Imaging of Signal Transduction Cascades with Probes Based on Förster Resonance Energy Transfer (FRET)
    12. UNIT 14.11 Biosensors for Characterizing the Dynamics of Rho Family GTPases in Living Cells
    13. UNIT 14.12 Analysis of Arf GTP-Binding Protein Function in Cells
    14. UNIT 14.13 Allosteric Activation of Kinases: Design and Application of RapR Kinases
    15. UNIT 14.14 Quantitative Analysis of Phosphoinositide 3-Kinase (PI3K) Signaling Using Live-Cell Total Internal Reflection Fluorescence (TIRF) Microscopy
    16. UNIT 14.15 A High-Content Assay for Biosensor Validation and for Examining Stimuli that Affect Biosensor Activity
  18. Chapter 15 Protein Trafficking
    1. Introduction
    2. UNIT 15.1 Overview of Protein Trafficking in the Secretory and Endocytic Pathways
    3. UNIT 15.2 Use of Glycosidases to Study Protein Trafficking
    4. UNIT 15.3 Endocytosis: Biochemical Analyses
    5. UNIT 15.4 Determining Protein Transport to the Plasma Membrane
    6. UNIT 15.5 Analysis of Membrane Traffic in Polarized Epithelial Cells
    7. UNIT 15.6 Analysis of Protein Folding and Oxidation in the Endoplasmic Reticulum
    8. UNIT 15.7 Measurements of Phagocytosis and Phagosomal Maturation
    9. UNIT 15.8 Analysis of Protein Transport to Lysosomes
    10. UNIT 15.9 Studies of the Ubiquitin Proteasome System
    11. UNIT 15.10 Measuring Retrograde Transport to the Trans-Golgi Network
    12. UNIT 15.11 Assays for Regulated Exocytosis of Mast Cell Granules
    13. UNIT 15.12 Analysis of Regulated Secretion Using PC12 Cells
    14. UNIT 15.13 Analysis of Endocytic Trafficking by Single-Cell Fluorescence Ratio Imaging
    15. UNIT 15.14 Quantitative Analysis of Endocytosis and Turnover of Epidermal Growth Factor (EGF) and EGF Receptor
    16. UNIT 15.15 Documenting GLUT4 Exocytosis and Endocytosis in Muscle Cell Monolayers
    17. UNIT 15.16 Measurement of Autophagic Activity in Mammalian Cells
    18. UNIT 15.17 Analysis of Polarized Membrane Traffic in Hepatocytes and Hepatic Cell Lines
    19. UNIT 15.18 Analysis of Rab GTPases
    20. UNIT 15.19 Synchronizing Protein Transport in the Secretory Pathway
    21. UNIT 15.20 Rapid Inactivation of Proteins by Knocksideways
  19. Chapter 16 Antibodies as Cell Biological Tools
    1. Introduction
    2. UNIT 16.1 Production of Monoclonal Antibodies
    3. UNIT 16.2 Production of Polyclonal Antisera
    4. UNIT 16.3 Purification of Immunoglobulin G
    5. UNIT 16.4 Fragmentation of Immunoglobulin G
    6. UNIT 16.5 Antibody Conjugates for Cell Biology
    7. UNIT 16.6 Production of Antibodies That Recognize Specific Tyrosine-Phosphorylated Peptides
  20. Chapter 17 Macromolecular Interactions in Cells
    1. Introduction
    2. UNIT 17.1 Imaging Protein-Protein Interactions by Fluorescence Resonance Energy Transfer (FRET) Microscopy
    3. UNIT 17.2 Identification of Protein Interactions by Far Western Analysis
    4. UNIT 17.3 Interaction Trap/Two-Hybrid System to Identify Interacting Proteins
    5. UNIT 17.4 Mapping Protein-Protein Interactions with Phage-Displayed Combinatorial Peptide Libraries
    6. UNIT 17.5 Protein-Protein Interactions Identified by Pull-Down Experiments and Mass Spectrometry
    7. UNIT 17.6 Measuring Protein Interactions by Optical Biosensors
    8. UNIT 17.7 Chromatin Immunoprecipitation for Determining the Association of Proteins with Specific Genomic Sequences In Vivo
    9. UNIT 17.8 Isothermal Titration Calorimetry
    10. UNIT 17.9 Rational Design and Evaluation of FRET Experiments to Measure Protein Proximities in Cells
    11. UNIT 17.10 Identification and Analysis of Multiprotein Complexes Through Chemical Crosslinking
    12. UNIT 17.11 Visualization of RNA Using Fluorescence Complementation Triggered by Aptamer-Protein Interactions (RFAP) in Live Bacterial Cells
    13. UNIT 17.12 Use of In Vivo Biotinylation for Chromatin Immunoprecipitation
    14. UNIT 17.13 Approaches to Studying Arf GAPs in Cells: In Vitro Assay with Isolated Focal Adhesions
    15. UNIT 17.14 Chromatin Immunoprecipitation of Adult Murine Cardiomyocytes
    16. UNIT 17.15 Identification of Genes Important for the Physical Interaction between Protein Pairs through Reverse PCA (rPCA)
    17. UNIT 17.16 Tools for Controlling Protein Interactions Using Light
    18. UNIT 17.17 Visualizing G Protein-Coupled Receptor-Receptor Interactions in Brain Using Proximity Ligation In Situ Assay
  21. Chapter 18 Cellular Aging and Death
    1. Introduction
    2. UNIT 18.1 Current Concepts in Cell Death
    3. UNIT 18.2 Analysis of Caspase Activation During Apoptosis
    4. UNIT 18.3 Assessment of Apoptosis and Necrosis by DNA Fragmentation and Morphological Criteria
    5. UNIT 18.4 Quantitative Fluorescence In Situ Hybridization (Q-FISH)
    6. UNIT 18.5 Analysis of Mitochondrial Dysfunction During Cell Death
    7. UNIT 18.6 Analysis of Telomeres and Telomerase
    8. UNIT 18.7 Nonisotopic Methods for Determination of Poly(ADP-Ribose) Levels and Detection of Poly(ADP-Ribose) Polymerase
    9. UNIT 18.8 Flow Cytometry of Apoptosis
    10. UNIT 18.9 Analysis of Cellular Senescence in Culture In Vivo: The Senescence-Associated β-Galactosidase Assay
    11. UNIT 18.10 High-Throughput Live Cell Imaging of Apoptosis
  22. Chapter 19 Whole Organism and Tissue Analysis
    1. Introduction
    2. UNIT 19.1 Overview of Metastasis Assays
    3. UNIT 19.2 Tail Vein Assay of Cancer Metastasis
    4. UNIT 19.3 Microanalysis of Gene Expression in Tissues Using T7-SAGE: Serial Analysis of Gene Expression After High-Fidelity T7-Based RNA Amplification
    5. UNIT 19.4 SAGE Analysis from 1 µg of Total RNA
    6. UNIT 19.5 The Chick Chorioallantoic Membrane as an In Vivo Angiogenesis Model
    7. UNIT 19.6 Experimental Metastasis Assays in the Chick Embryo
    8. UNIT 19.7 Imaging Tumor Cell Movement In Vivo
    9. UNIT 19.8 Embryonic Organ Culture
    10. UNIT 19.9 Three-Dimensional Tissue Models of Normal and Diseased Skin
    11. UNIT 19.10 Overview: Engineering Transgenic Constructs and Mice
    12. UNIT 19.11 Generation of Transgenic Mice
    13. UNIT 19.12 Overview: Generation of Gene Knockout Mice
    14. UNIT 19.13 Manipulation of Mouse Embryonic Stem Cells for Knockout Mouse Production
    15. UNIT 19.14 Generation of Gene Knockout Mice by ES Cell Microinjection
    16. UNIT 19.15 Chick Embryo Culture and Electroporation
    17. UNIT 19.16 3-D Extracellular Matrix from Sectioned Human Tissues
    18. UNIT 19.17 Reconstitution of a Bioengineered Salivary Gland Using a Three-Dimensional Cell Manipulation Method
    19. UNIT 19.18 Analysis of the Stromal Cellular Components of the Solid Tumor Microenvironment Using Flow Cytometry
  23. Chapter 20 Expression and Introduction of Macromolecules into Cells
    1. Introduction
    2. UNIT 20.1 Direct Introduction of Molecules into Cells
    3. UNIT 20.2 Protein Transduction: Generation of Full-Length Transducible Proteins Using the TAT System
    4. UNIT 20.3 Calcium Phosphate Transfection
    5. UNIT 20.4 Transfection Using DEAE-Dextran
    6. UNIT 20.5 Transfection by Electroporation
    7. UNIT 20.6 Transfection of Cultured Eukaryotic Cells Using Cationic Lipid Reagents
    8. UNIT 20.7 Optimization of Transfection
    9. UNIT 20.8 Inducible Gene Expression Using an Autoregulatory, Tetracycline-Controlled System
    10. UNIT 20.9 Rapid Depletion of Budding Yeast Proteins via the Fusion of an Auxin-Inducible Degron (AID)
  24. Chapter 21 Fluorescent Protein Technology
    1. Introduction
    2. UNIT 21.1 Measuring Protein Mobility by Photobleaching GFP Chimeras in Living Cells
    3. UNIT 21.2 Fluorescence Localization After Photobleaching (FLAP)
    4. UNIT 21.3 Visualization of Protein Interactions in Living Cells Using Bimolecular Fluorescence Complementation (BiFC) Analysis
    5. UNIT 21.4 Design and Use of Fluorescent Fusion Proteins in Cell Biology
    6. UNIT 21.5 The Fluorescent Protein Color Palette
    7. UNIT 21.6 Photoactivation and Imaging of Photoactivatable Fluorescent Proteins
    8. UNIT 21.7 Probing Endoplasmic Reticulum Dynamics using Fluorescence Imaging and Photobleaching Techniques
    9. UNIT 21.8 Superresolution Imaging with Standard Fluorescent Probes
    10. UNIT 21.9 Probing Peroxisome Dynamics and Biogenesis by Fluorescence Imaging
  25. Chapter 22 Cell Biology of Chromosomes and Nuclei
    1. Introduction
    2. UNIT 22.1 Overview of Cytogenetic Chromosome Analysis
    3. UNIT 22.2 Preparation of Cytogenetic Specimens from Tissue Samples
    4. UNIT 22.3 Traditional Banding of Chromosomes for Cytogenetic Analysis
    5. UNIT 22.4 Fluorescence In Situ Hybridization (FISH)
    6. UNIT 22.5 Multi-Color FISH Techniques
    7. UNIT 22.6 Comparative Genomic Hybridization
    8. UNIT 22.7 Sister Chromatid Exchange
    9. UNIT 22.8 Detection of Mitotic Figures and Components of the Mitotic Machinery
    10. UNIT 22.9 Assembly and Micromanipulation of Xenopus In Vitro–Assembled Mitotic Chromosomes
    11. UNIT 22.10 Replication Labeling with Halogenated Thymidine Analogs
    12. UNIT 22.11 Assays for Ribosomal RNA Processing and Ribosome Assembly
    13. UNIT 22.12 Visualization and Measurement of DNA Methyltransferase Activity in Living Cells
    14. UNIT 22.13 Monitoring mRNA Export
    15. UNIT 22.14 Analysis of DNA Replication in Saccharomyces cerevisiae by Two-Dimensional and Pulsed-Field Gel Electrophoresis
    16. UNIT 22.15 Analysis of Interactions Between Genomic Loci Through Chromosome Conformation Capture (3C)
    17. UNIT 22.16 Assays to Measure Nuclear Mechanics in Interphase Cells
    18. UNIT 22.17 Scoring and Manipulating Gene Position and Dynamics Using FROS in Budding Yeast
    19. UNIT 22.18 Best Practices for Mapping Replication Origins in Eukaryotic Chromosomes
    20. UNIT 22.19 Analysis of Copy-Number Alterations in Single Cells Using Microarray-Based Comparative Genomic Hybridization (aCGH)
    21. UNIT 22.20 Mapping Replication Origin Sequences in Eukaryotic Chromosomes
  26. Chapter 23 Stem Cells
    1. Introduction
    2. UNIT 23.1 Stem Cells: An Overview
    3. UNIT 23.2 Mouse Embryonic Stem Cell Derivation, and Mouse and Human Embryonic Stem Cell Culture and Differentiation as Embryoid Bodies
    4. UNIT 23.3 Maintenance and In Vitro Differentiation of Mouse Embryonic Stem Cells to Form Blood Vessels
    5. UNIT 23.4 Differentiation of Mouse Embryonic Stem Cells and of Human Adult Stem Cells into Adipocytes
    6. UNIT 23.5 Induction of ES Cell–Derived Cartilage Formation
    7. UNIT 23.6 Hematoendothelial Differentiation of Human Embryonic Stem Cells
    8. UNIT 23.7 Neural Differentiation of Human ES Cells
    9. UNIT 23.8 Cardiac Differentiation of Human Embryonic Stem Cells and their Assembly into Engineered Heart Muscle
    10. UNIT 23.9 Expanding Mouse Ventricular Cardiomyocytes Through GSK-3 Inhibition
    11. UNIT 23.10 Isolation and Culturing of Glioma Cancer Stem Cells
  27. Chapter 24 Lipids
    1. Introduction
    2. UNIT 24.1 Using Fluorescent Sphingolipid Analogs to Study Intracellular Lipid Trafficking
    3. UNIT 24.2 Fluorescent Detection of Lipid Droplets and Associated Proteins
    4. UNIT 24.3 Making Giant Unilamellar Vesicles via Hydration of a Lipid Film
    5. UNIT 24.4 Visualization of Cellular Phosphoinositide Pools with GFP-Fused Protein-Domains
    6. UNIT 24.5 Supported Lipid Bilayer Technology for the Study of Cellular Interfaces
  28. Chapter 25 Nanotechnology
    1. Introduction
    2. UNIT 25.1 In Vivo Imaging Using Quantum Dot–Conjugated Probes
    3. UNIT 25.2 Fabrication and Application of Nanofibrous Scaffolds in Tissue Engineering
    4. UNIT 25.3 Three-Dimensional Patterning of the ECM Microenvironment Using Magnetic Nanoparticle Self Assembly
  29. Chapter 26 Viruses
    1. Introduction
    2. UNIT 26.1 Production of Papillomavirus-Based Gene Transfer Vectors
    3. UNIT 26.2 BK Virus (BKV): Infection, Propagation, Quantitation, Purification, Labeling, and Analysis of Cell Entry
    4. UNIT 26.3 Methods Used to Study Respiratory Virus Infection
    5. UNIT 26.4 Compartmented Neuron Cultures for Directional Infection by Alpha Herpesviruses
    6. UNIT 26.5 HIV-1 Interactions with Cells: From Viral Binding to Cell-Cell Transmission
    7. 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
    8. UNIT 26.7 COS-1 Cells as Packaging Host for Production of Lentiviruses
    9. UNIT 26.8 Production of Lentiviral Vectors in Protein-free Media
    10. UNIT 26.9 Monitoring Viral-Mediated Membrane Fusion Using Fluorescent Reporter Methods
    11. UNIT 26.10 Evaluation of Antiseptic Antiviral Activity of Chemical Agents
    12. UNIT 26.11 Rapid Titration of Viruses by Flow Cytometry
    13. UNIT 26.12 An Enzymatic Assay for Detection of Viral Entry
    14. UNIT 26.13 A Dual-Chamber Model of the Female Genital Tract to Evaluate Epithelial Toxicity of Candidate Anti-HIV Microbicides
    15. UNIT 26.14 Methods for Growing and Titrating African Swine Fever Virus: Field and Laboratory Samples
  30. Chapter 27 RNA-Based Methods in Cell Biology
    1. Introduction
    2. UNIT 27.1 Silencing of Gene Expression in Cultured Cells Using Small Interfering RNAs
    3. UNIT 27.2 Gene Down-Regulation with Short Hairpin RNAs and Validation of Specificity by Inducible Rescue in Mammalian Cells
    4. UNIT 27.3 Analysis of Nonsense-Mediated mRNA Decay in Saccharomyces cerevisiae
    5. UNIT 27.4 Analysis of Nonsense-Mediated mRNA Decay in Mammalian Cells
  31. Appendix 1 Useful Information and Data
    1. APPENDIX 1A Useful Measurements and Data
    2. APPENDIX 1B Compendium of Drugs Commonly Used in Cell Biology Research
    3. APPENDIX 1C Identification of Motifs in Protein Sequences
    4. APPENDIX 1D Safe Use of Radioisotopes
    5. APPENDIX 1E Absorption and Emission Maxima for Common Fluorophores
    6. APPENDIX 1F Importing Biological Materials
    7. APPENDIX 1G Centrifuges and Rotors
    8. APPENDIX 1H Internet Basics for Biologists
  32. Appendix 2 Laboratory Stock Solutions and Equipment
    1. APPENDIX 2A Common Stock Solutions, Buffers, and Media
    2. APPENDIX 2B Medium Formulations
    3. APPENDIX 2C Standard Laboratory Equipment
  33. Appendix 3 Commonly Used Techniques
    1. APPENDIX 3A Molecular Biology Techniques
    2. APPENDIX 3B Spectrophotometric Determination of Protein Concentration
    3. APPENDIX 3C Dialysis and Concentration of Protein Solutions
    4. APPENDIX 3D Quantification of DNA and RNA with Absorption and Fluorescence Spectroscopy
    5. APPENDIX 3E Silanizing Glassware
    6. APPENDIX 3F Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization
    7. APPENDIX 3G Micro RT-PCR
    8. APPENDIX 3H The Colorimetric Detection and Quantitation of Total Protein
  34. Chapter 3 Commonly Used Techniques
    1. APPENDIX A.3I Standardized Cryopreservation of Human Primary Cells