Current Protocols in Cell Biology
Table of Contents
Foreword by Kai
Simons
Preface
1 Cell Culture
1.1 Basic Techniques for Mammalian Cell Tissue Culture
1.2 Media for Culture of Mammalian Cells
1.3 Aseptic Technique for Cell Culture
1.4 Sterilization and Filtration
1.5 Assessing and Controlling Microbial Contamination in Cell Cultures
1.6 Media and Culture of Yeast
1.7 BY-2 Cells: Culture and Transformation for Live Cell Imaging
New, June 2003)
1.7 BY-2 Cells: Culture and Transformation for Live Cell Imaging
2 Preparation and Isolation of Cells
2.1 Establishment of Fibroblast Cultures
2.2 Preparation and Culture of Human Lymphocytes
2.3 Preparation of Endothelial Cells
2.4 Generation of Continuously Growing B Cell Lines by Epstein-Barr
Virus Transformation
2.5 Laser Capture Microdissection
2.6 Preparation of Human Epidermal Keratinocyte Cultures
2.7 Preparation and Coculture of Neurons and Glial Cells
3 Subcellular Fractionation and Isolation of Organelles
3.1 Overview of Cell Fractionation
3.2 Isolation of Rat Hepatocyte Plasma Membrane Sheets and Plasma
Membrane Domains
3.3 Isolation of Mitochondria from Tissues and Cells by Differential
Centrifugation
3.4 Purification of a Crude Mitochondrial Fraction by Density-Gradient
Centrifugation
3.5 Isolation of Peroxisomes from Tissues and Cells by Differential
and Density Gradient Centrifugation
3.6 Isolation of Lysosomes from Tissues and Cells by Differential
and Density Gradient Centrifugation
3.7 Overview of Subcellular Fractionation Procedures for the Yeast
Saccharomyces cerevisiae
3.8 Isolation of Subcellular Fractions from the Yeast Saccharomyces
cerevisiae
3.9 Isolation of Golgi Membranes from Tissues and Cells by Differential
and Density Gradient Centrifugation
3.10 Isolation of Nuclei and Nuclear Membranes from Animal Tissues
3.11 Free-Flow Electrophoretic Analysis of Endosome Subpopulations
of Rat Hepatocytes
3.12 Isolation of Synaptic Vesicles
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
4 Microscopy
4.1 Proper Alignment and Adjustment of the Light Microscopes
4.2 Fluorescence Microscopy
4.3 Immunofluorescence Staining
4.4 Fluorescent Staining of Subcellular Organelles: ER, Golgi Complex,
and Mitochondria
4.5 Basic Confocal Microscopy
4.6 Immunoperoxidase Methods for Localization of Antigens in Cultured
Cells and Tissues
4.7 Cryo-Immunogold Electron Microscopy
4.8 Correlative Video Light/Electron Microscopy
4.9 Polarization Microscopy
4.10 Fluorescent Speckle Microscopy (FSM) of Microtubules and Actin
in Living Cells
4.11 Two-Photon Excitation Microscopy for the Study of Living Cells
and Tissues
4.12 Total Internal Reflection Fluorescence Microscopy for High-Resolution
Imaging of Cell-Surface Events
4.13 Fluorescent Labeling of Yeast
4.14 Fluorescence Lifetime Imaging Microscopy
4A Organelle Atlas: Appendix to Chapter 4
4A Organelle Atlas
This atlas contains images of cellular organelles visualized using
a wide variety of reagents and techniques and demonstrates the diversity
of methods for exploring the cell.
5 Characterization of Cellular Proteins
5.1 Overview of Physical State of Proteins Within Cells
5.2 Determining the Topology of an Integral Membrane Protein
5.3 Determination of Molecular Size by Zonal Sedimentation Analysis
on Sucrose Density Gradients
5.4 Analysis of the Association of Proteins with Membranes
5.5 Determination of Molecular Size by Size-Exclusion Chromatography
(Gel Filtration)
5.6 Identification of Proteins in Complex Mixtures Using Liquid
Chromatography and Mass Spectrometry
6 Electrophoresis and Immunoblotting
6.1 One-Dimensional SDS Gel Electrophoresis of Proteins
6.2 Immunoblotting and Immunodetection
6.3 Detection and Quantitation of Radiolabeled Proteins in Gels
6.4 Two-Dimensional Gel Electrophoresis
6.5 One-Dimensional Electrophoresis Using Nondenaturing Conditions
6.6 Staining Proteins in Gels
6.7 Agarose Gel Electrophoresis of Proteins
6.8 Fluorescence Detection of Glycoproteins in Gels and on Electroblots
6.9 Digital Electrophoresis Analysis
7 Protein Labeling and Immunoprecipitation
7.1 Metabolic Labeling with Amino Acids
7.2 Immunoprecipitation
7.3 Metabolic Labeling with Sulfate
7.4 Metabolic Labeling with Fatty Acids
7.5 Metabolic Labeling of Prenyl and Carboxyl-Methyl Groups
7.6 Metabolic Labeling and Immunoprecipitation of Yeast Proteins
7.7 Metabolic Labeling and Immunoprecipiation of Drosophila
Proteins
7.8 Metabolic Labeling of Glycoproteins with Radioactive Sugars
7.9 Analysis of Oxidative Modification of Proteins
7.10 Radioiodination of Cellular Proteins
8 Cell Cycle Analysis
8.1 Overview of the Cell Cycle
8.2 Assays for CDK Activity and DNA Replication in the Cell Cycle
8.3 Methods for Synchronizing Cells at Specific Stages of the Cell
Cycle
8.4 Determining Cell Cycle Stages by Flow Cytometry
8.5 Centrifugal Elutriation to Obtain Synchronous Populations of
Cells
9 Cell Adhesion
9.1 Cell-Substrate Adhesion Assays
9.2 Quantitative Measurement of Cell Adhesion Using Centrifugal
Force
9.3 Cadherin-Dependent Cell-Cell Adhesion
9.4 Analyzing Integrin-Dependent Adhesion
9.5 Analysis of Cell-Cell Contact Mediated by Ig Superfamily Cell
Adhesion Molecules
9.6 Measurement of Adhesion Under Flow Conditions(New, March 2003)
10 Extracellular Matrix
10.1 Overview of Extracellular Matrix
10.2 Preparation of Basement Membrane Components from EHS Tumors
10.3 Preparation of Gelled Substrates
10.4 Preparation of Extracellular Matrices Produced by Cultured
Cells
10.5 Purification of Fibronectin
10.6 Purification of Vitronectin
10.7 Proteoglycan Isolation and Analysis
10.8 Matrix Metalloproteinases
10.9 Preparation of Extracellular Matrices Produced by Cultured
Fibroblasts
10.10 Purification and Analysis of Thrombospondin-1
10.11 Purification of SPARC/Osteonectin
10.12 Analysis of Fibronectin Matrix Assembly
10.13 Non-Radioactive Quantification of Fibronectin Matrix Assembly
11 In Vitro Reconstitution
11.1 Overview of Eukaryotic In Vitro Translation and Expression
Systems
11.2 In Vitro Translation
11.3 In Vitro Analysis of Endoplasmic-Reticulum-to-Golgi Transport
in Mammalian Cells
11.4 Cotranslational Translocation of Proteins into Canine Rough
Microsomes
11.5 In Vitro Analysis of SV40 DNA Replication
11.6 In Vitro Transcription
11.7 Nuclear Import in Digitonin-Permeabilized Cells
11.8 In Vitro Translation Using HeLa Extract
11.9 Analysis of Eukaryotic Translation in Purified and Semipurified
Systems
11.10 Preparation and Use of Interphase Xenopus Egg Extracts
11.11 Analysis of the Cell Cycle Using Xenopus Egg Extracts
11.12 Analysis of Apoptosis Using Xenopus Egg Extracts
11.13 Mitotic Spindle Assembly In Vitro
11.14 Analysis of RNA Export Using Xenopus Oocytes
11.15 In Vitro Analysis of Peroxisomal Protein Import
11.16 In Vitro Analysis of Chloroplast Protein Import
11.17 In Vitro RNA Splicing in Mammalian Cell Extracts(New,
March 2003)
11.18 Endocytosis Assays in Intact and Permeabilized Cells
12 Cell Motility
12.1 Chemotaxis Assays for Eukaryotic Cells
12.2 Invasion Assays
12.3 Cell Traction
12.4 Cell Wounds Assays
12.5 Dictyostelium Cell Dynamics
12.6 Optical Microscopy-Based Migration Assay for Human Neutrophils
12.7 Actin-Based Motility Assay
12.8 In Vivo Marking of Single Cells in Chick Embryos Using Photoactivation of GFP
13 Organelle Motility
13.1 Microtubule-Organelle Motility Assays
13.2 In Vitro Assay to Study Translocation of Actin by Myosin
13.3 Organelle Motility in Plant Cells: Imaging Golgi and ER Dynamics
with GFP
13.4 Movement of Nuclei
13.5 Measuring Dynamics of Nuclear Proteins by Photobleaching(New,
March 2003)
13.6 Functional Characterization of Proteins Regulating Actin Assembly
14 Signal Transduction: Protein Phosphorylation
14.1 Overview of Protein Phosphorylation
14.2 Immunological Detection of Phosphorylation
14.3 Detection of MAP Kinase Signaling
14.4 Labeling Cultured Cells with 32Pi and
Preparing Cell Lysates for Immunoprecipitation
14.5 Phosphoamino Acid Analysis
14.6 Determination of Akt/PKB Signaling
14.7 Analyzing FAK and Pyk2 in Early Integrin Signaling Events
15 Protein Trafficking
15.1 Overview of Protein Trafficking in the Secretory and Endocytic
Pathways
15.2 Use of Glycosidases to Study Protein Trafficking
15.3 Endocytosis: Biochemical Analyses
15.4 Determining Protein Transport to the Plasma Membrane
15.5 Analysis of Membrane Traffic in Polarized Epithelial
Cells
15.6 Analysis of Protein Folding and Oxidation in the Endoplasmic
Reticulum
15.7 Measurements of Phagocytosis and Phagosomal Maturation
5.8 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
16 Antibodies as Cell Biological Tools
16.1 Production of Monoclonal Antibodies
16.2 Production of Polyclonal Antisera
16.3 Purification of Immunoglobulin G
16.4 Fragmentation of Immunoglobulin G
16.5 Antibody Conjugates for Cell Biology
16.6 Production of Antibodies That Recognize Specific Tyrosine-Phosphorylated
Peptides
17 Macromolecular Interaction in Cells
17.1 Imaging Protein-Protein Interactions by Fluorescence Resonance
Energy Transfer (FRET) Microscopy
17.2 Identification of Protein Interactions by Far Western Analysis
17.3 Interaction Trap/Two-Hybrid System to Identify Interacting
Proteins
17.4 Mapping Protein-Protein Interactions with Phage-Displayed Combinatorial
Peptide Libraries
17.5 Protein-Protein Interactions Identified by Pull-Down Experiments
and Mass Spectrometry
17.6 Measuring Protein Interactions by Optical Biosensors
17.7 Chromatin Immunoprecipitation for Determining the Association
of Proteins with Specific Genomic Sequences In Vivo HTML PDF (Size
240KB)
17.8 Isothermal Titration Calorimetry
17.9 Rational Design and Evaluation of FRET Experiments to Measure Protein Proximities in Cells
18 Cellular Aging and Death
18.1 Current Concepts in Cell Death
18.2 Analysis of Caspase Activation During Apoptosis
18.3 Assessment of Apoptosis and Necrosis by DNA Fragmentation and
Morphological Criteria )
18.4 Quantitative Fluorescence In Situ Hybridization (Q-FISH)
18.5 Analysis of Mitochondrial Dysfunction During Cell Death <New,
June 2003)
18.6 Analysis of Telomeres and Telomerase HTML PDF (Size 647KB)
U18.7 Nonisotopic Methods for Determination of Poly(ADP-Ribose)
Levels and Detection of Poly(ADP-Ribose) Polymerase )
18.8 Flow Cytometry of Apoptosis
18.9 Analysis of Cellular Senescence in Culture In Vivo: The Senescence-Associated  -Galactosidase Assay
19 Whole Organism and Tissue Analyses
19.1 Overview of Metastasis Assays
19.2 Tail Vein Assay of Cancer Metastasis
19.3 Microanalysis of Gene Expression in Tissues Using T7-SAGE:
Serial Analysis of Gene Expression After High-Fidelity T7-Based
RNA Amplification
19.4 SAGE Analysis from 1 mg of Total RNA
19.5 The Chick Chorioallantoic Membrane As an In Vivo Angiogenesis
Model (New, March 2003)
19.6 Experimental Metastasis Assays in the Chick Embryo
20 Expression and Introduction of Macromolecules
in Cells
20.1 Direct Introduction of Molecules into Cells (New, March
2003)
20.2 Protein Transduction: Generation of Full-Length Transducible
Proteins Using the TAT System (New, March 2003)
20.3 Calcium Phosphate Transfection New, June 2003)
20.4 Transfection Using DEAE-Dextran New, June 2003)
20.5 Transfection by Electroporation New, June 2003)
20.6 Transfection of Cultured Eukaryotic Cells Using Cationic
Lipid Reagents New, June 2003)
20.7 Optimization of Transfection New, June 2003)
20.8 Inducible Gene Expression Using an Autoregulatory, Tetracycline-Controlled System
21 Fluorescent Protein Technology
Introduction
21.2 Fluorescence Localization After Photobleaching (FLAP)
21.1 Measuring Protein Mobility by Photobleaching GFP Chimeras
in Living Cells
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
22 Cell Biology of Chromosomes and Nuclei
Introduction HTML PDF (Size 28KB)
22.1 Overview of Cytogenetic Chromosome Analysis
22.2 Preparation of Cytogenetic Specimens from Tissue Samples
22.3 Traditional Banding of Chromosomes for Cytogenetic Analysis)
22.4 Fluorescence In Situ Hybridization (FISH)
22.5 Multi-Color FISH Techniques
22.6 Comparative Genomic Hybridization
22.7 Sister Chromatid Exchange
22.8 Detection of Mitotic Figures and Components of the Mitotic
Machinery
22.9 Assembly and Micromanipulation of Xenopus In Vitro—Assembled Mitotic Chromosomes
Chapter 23 Stem Cells
Introduction
Unit 23.1 Stem Cells: An Overview
23.2 Mouse Embryonic Stem Cell Derivation, and Mouse and Human Embryonic Stem Cell Culture and Differentiation as Embryoid Bodies
Appendices
A.1 USEFUL INFORMATION AND DATA
A. Useful Measurements and Data
B. Compendium of Drugs Commonly
Used in Cell Biology
C. Identification of Motifs
in Protein Sequences
D. Safe Use of Radioisotopes
E. Absorption and Emission Maxima
of Common Fluorophores
F. Importing Biological Materials
G. Centrifuges and Rotors
H Internet Basics for Biologists
A.2 LABORATORY STOCK SOLUTIONS AND EQUIPMENT
A. Common Stock Solutions,
Buffers, and Media
B. Media Formulations
C. Standard Laboratory Equipment
A.3 COMMONLY USED TECHNIQUES
A. Molecular Biology Techniques
B. Spectrophotometric Determination
of Protein Concentration
C. Dialysis and Concentration
of Protein Solutions
D. Quantification of DNA and
RNA with Absorption and Fluorescence Spectroscopy
E. Silanizing Glassware
F. Enzymatic Amplification
of DNA by PCR: Standard Procedures and Optimization
G. Micro RT-PCR
H. The Colorimetric Detection
and Quantitation of Total Protein
SUPPLIERS APPENDIX
Selected Suppliers of Reagents and Equipment
Index
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