Cell Biology

Part 1: Tissue Culture and Associated Techniques

Section A: General Techniques

General Procedures for Tissue Culture

Development of Serum-Free Media and Methods for Optimization of Nutrient Composition

Testing Cell Cultures for Microbial and Viral Contaminants

Section B: Primary Cultures from Embryonic Tissues

Primary and Extended Culture of Embryonic Mouse Cells: Establishment of a Novel Cell Culture Model of Apoptosis and Neural Differentiation

Tissue Culture of Embryonic Stem Cells

Isolation and Culture of Germ Cells from the Mouse Embryo

Section C: Cultures of Specific Cell Types

Epithelial Cells

Cultivation of Human Epidermal Keratinocytes with a 3T3 Feeder Layer

Growth of Human Keratinocytes in Serum-Free Medium

Isolation of Hepatocytes

Isolation and Culture of Oval Cells from Carcinogen-Treated Rats

In Vitro Culture of Mouse Fetal Choroid Plexus Epithelial Cells

Isolation and Culture of Type II Pulmonary Epithelial Cells

Mesenchymal Cells

Maintenance of Human Diploid Fibroblast-Like Cells in Culture

Isolation of Osteoclasts and Osteoclast Plasma Membranes

Culturing of Human Umbilical Vein and Dermal Microvascular Endothelial Cells

Neuroectodermal Cells

Isolation and Proliferation of Adult Mammalian Central Nervous System Stem Cells

Hemopoietic Cells

Clonal Cultures in Vitro for Hemopoietic Cells Using Semisolid Agar Medium

Gonads

Properties of Isolated Sertoli Cells

Culture of Ovarian Granulosa Cells: Calcium Imaging at the Single-Cell Level

Section D: Cell Separation Techniques

Isolation of Peripheral Blood Mononuclear Cells and Identification of Human Lymphocyte Subpopulations by Multiparameter Flow Cytometry

Purification of Functionally Active Epidermal Langerhans Cells Using Immunomagnetic Beads

Section E: Model Systems to Study Differentiation

Nonterminal and Terminal Adipocyte Differentiation of Murine 3T3 T Mesenchymal Stem Cells

Cell Systems for ex Vivo Studies of Myogenesis: A Protocol for the Isolation of Stable Muscle Cell Populations from Newborn to Adult Mice

Induction of Cell Differentiation in Human HL-60 Promyelocyte Leukemia Cells: Quantitation of a Myeloid Specific Antigen, MRP-8/MRP-14 Protein Complex

Differentiation of Murine Erythroleukemia Cells (Friend Cells)

Cultured PC 12 Cells: A Model for Neuronal Function and Differentiation

Growing Madin-Darby Canine Kidney Cells for Studying Epithelial Cell Biology

In Vitro Studies of Epithelium-to-Mesenchyme Transitions

Section F: Immortalization of Cells

Inducible Immortalization of Cells from Transgenic Mice Expressing Simian Virus 40 under lac Operon Control

Immortalization of Rat Ventral Prostate Epithelial Cells Using Simian Virus 40 T Antigen

Section G: Cell Cycle Analysis

Cell Cycle Analysis by Flow Cytometry

Preparation of Synchronous Populations of Mammalian Cells in Specific Phases of the Cell Cycle by Centrifugal Elutriation

Synchronization of Normal Diploid and Transformed Mammalian Cells

Synchronization of Transformed Human Amnion Cells by Mitotic Detachment

Stimulation of DNA Synthesis in Quiescent 3T3 Cells

Section H: Cytotoxic Assays

Quantitative Determination of Compound Cytotoxicity in Proliferating Cells: Monitoring DNA Synthesis by [3H]Thymidine Incorporation

Section I: Senescence, Programmed Cell Death, and Others

Serial Propagation of Human Fibroblasts for the Study of Aging at the Cellular Level

Morphological Criteria for Identifying Apoptosis

Use of the Terminal Transferase DNA Labeling Reaction for the Biochemical and in Situ Analysis of Apoptosis

Growth and Induction of Metastasis of Mammary Epithelial Cells

Measurement of Cell-Cell and Cell-Extracellular Matrix Interactions: A Quantitative Cell Attachment Assay

Section J: Electrophysiological Methods

Patch-Clamp Recording

Section K: Histocultures

Three-Dimensional Sponge-Gel Matrix Histoculture: Methods and Applications

Section L: Other Cell Types

Anthropoda

Primary Culture of Drosophila Embryo Cells

Caenorhabditis elegans

Laboratory Cultivation of Caenorhabditis elegans and Other Free-Living Nematodes

Protozoa

Cultivation of Tetrahymena Cells

Acanthamoeba castellanii: A Model System for Correlative Biochemical and Cell Biological Studies

Fungi

Cell Biological, Molecular Genetic, and Biochemical Methods to Examine Dictyostelium

Large-Scale Culture of Physarum: A Simple Method for Growing Several Hundred Grams of Plasmodia

Plants

Induction of Regeneration-Competent Monocot Callus

Isolation, Culture, and Plant Regeneration from Protoplasts

Part 2: Viruses

Propagation and Purification of Polyoma and Simian Virus 40

Construction and Propagation of Human Adenovirus Vectors

Tissue Culture Techniques for the Study of Human Papilloma Viruses

Growth and Purification of Murine Leukemia Virus

Part 3: Organelles, Cellular Structures, Macromolecules, and Functional Assays

Purification of Rat Liver Golgi Stacks

Preparation and Purification of Post-Golgi Transport Vesicles from Perforated Madin-Darby Canine Kidney Cells

Purification of Clathrin-Coated Vesicles from Bovine Brain, Liver, and Adrenal Gland

Functional Identification of Membranes Derived from the Rough Endoplasmic Reticulum of Yeast

Isolation of Yeast Mitochondria and Study of Mitochondrial Protein Translation

Inclusion of Proteins into Isolated Mitochondrial Outer Membrane Vesicles

Isolation of Peroxisomes

Purification of Secretory Granules from PC 12 Cells

Preparation of Synaptic Vesicles from Mammalian Brain

Purification and Reconstitution of the Ca2+ -ATPase of Red Blood Cells

Isolation of Focal Adhesions from Cultured Cells

Isolation of Laminins from Tumor Sources and from Normal Tissues

Isolation of Centrosomes from Cultured Animal Cells

Preparation of Yeast Spindle Pole Bodies

Preparation of Nuclei and Nuclear Envelopes, Identification of an Integral Membrane Protein Unique to the Nuclear Envelope

Preparation of Cytoplasts and Karyoplasts from HeLa Cell Monolayers

Isolation and Visualization of the Nuclear Matrix, the Nonchromatin Structure of the Nucleus

Preparation of U Small Nuclear Ribonucleoprotein Particles

Rapid Preparation of hnRNP Core Proteins and Stepwise Assembly of hnRNP Particles in Vitro

Preparation of Ribosomes and Ribosomal Proteins from Cultured Cells

Preparation of Proteasomes

Small-Scale Preparation of Nuclear Extracts from Mammalian Cells

Purification of DNA Using Guanidine Thiocyanate and Isobutyl Alcohol Fractionation

Single-Step Method of Total RNA Isolation by Acid Guanidine-Phenol Extraction

Part 4: Microscopy Techniques

Section A: Light Microscopy

Phase-Contrast, Nomarski (Differential-Interference) Contrast, and Dark- Field Microscopy: Black and White and Color Photomicrography

Reflection Interference Microscopy

Using Interference Microscopy to Study Cell Behavior

Section B: Fluorescence Microscopy

Fluorescence Imaging in Living Cells

Practical Laser-Scanning Confocal Light Microscopy: Obtaining Optimal Performance from Your Instrument

Caged Fluorescent Probes for Monitoring Cytoskeleton Dynamics

Section C: Video Microscopy

Video-Enhanced Contrast Microscopy

Section D: Confocal Microscopy

Confocal Microscopy of Polarized MDCK Epithelial Cells

Real-Time Confocal Microscopy and Cell Biology

Section E: Electron Microscopy

Fixation of Cells and Tissues for Transmission Electron Microscopy

Embedding of Cells and Tissues for Ultrastructural and Immunocytochemical Analysis

Negative Staining

Whole-Mount Electron Microscopy of the Cytoskeleton: Negative Staining Methods

Glycerol Spraying/Low-Angle Rotary Metal Shadowing

Rapid Freezing of Biological Specimens for Freeze-Fracture and Deep Etching

Freeze Fracture and Freeze Etching

Use of Ultrathin Cryo- and Plastic Sections for Immunoelectron Microscopy

Cryo-Transmisson Electron Microscopy of Thin Vitrified Sections

Preparation Methods for Quantitative X-Ray Microanalysis of intracellular Elements in Ultrathin Sections for Transmission Electron Microscopy: The Freeze-Dry, Resin-Embed Route

High-Resolution Scanning Electron Microscopy in Cell Biology

Part 5: Microdissection Techniques

Microdissection-Based Techniques for the Determination of Cell Proliferation in Gastrointestinal Epithelium: Application to Animal and Human Studies

Micromanipulation of Chromosomes Using Laser Microsurgery (Optical Scissors) and Laser-Induced Optical Forces [Optical Tweezers)

Microdissection of Chromosomes and Microcloning

Part 6: Histochemistry

Mayer's Hematoxylin-Eosin: An Example of a Common Histological Staining Method

Selected Enzyme Staining in Histochemistry

Part 7: Antibodies

Section A: Production of Antibodies

Production of Polyclonal Antibodies in Rabbits

Production of Mouse Monoclonal Antibodies

Production of Human Monoclonal Antibodies via Fusion of Epstein-Barr Virus-Transformed Lymphocytes with Heteromyeloma

Rapid Production of Antibodies in Chicken and Isolation from Eggs

Section B: Purification and Labeling of Immunoglobulins

Purification of Immunoglobulins

Conjugation of Fluorescent Dyes to Antibodies

Section C: Antibody Specificity

Determination of Antibody Specificity by Western Blotting and Immunoprecipitation

Western Blotting and Ligand Blotting Using Enhanced Chemiluminescence and Radioiodine Detection

Enzyme-Linked Immunosorbent Assay

A Simple Solid-Phase Mutual Inhibition Assay Using Biotinylated Antigen for Analyzing the Epitope Specificities of Monoclonal Antibodies

DNA Immunoprecipitation: Application to Characterization of Target Sequences for a Human Centromere DNA-binding Protein (CENP-B)

Part 8: Immunocytochemistry And Vital Staining of Cells

Immunofluorescence Microscopy of Cultured Cells

Immunofluorescence Microscopy of the Cytoskeleton: Double and Triple Immunofluorescence

Immunocytochemistry of Frozen and of Paraffin Tissue Sections

Fluorescent Labeling of Nascent RNA in the Cell Nucleus Using 5-Bromouridine 5'-Triphosphate

Labeling of Endocytic Vesicles Using Fluorescent Probes for Fluid-Phase Endocytosis

Labeling of the Endoplasmic Reticulum with DiO6 (3)

Use of Fluorescent Analogs of Ceramide to Study the Golgi Apparatus of Animal Cells

Vital Staining of Mitochondria with Rhodamine 123 and of Acidic Organelles with Acridine Orange

Part 9: Intracellular Measurements

Measuring Membrane Potential in Single Cells with Confocal Microscopy

Measurement of Cytosolic pH in Single Cells by Dual-Excitation Fluorescence Spectrometry: Simultaneous Visualization Using Hoffman Modulation Contrast Optics

Part 10: Cytogenetics and in Situ Hybridization

Basic Cytogenetic Techniques: Culturing, Slide Making, and G-Banding

Production of Viable Hybrids Between Adherent Cells

Microcell-Mediated Chromosome Transfer: Selective Transfer and Retention of Single Human Chromosomes into Recipient Cells of Choice

Microcell Transfer of Chromosomes from Mitotic Cells

Chromosome Painting Using Degenerate Oligonucleotide-Primed Polymerase Chain Reaction-Amplified, Flow-Sorted Human Chromosomes

Fluorescence in Situ Hybridization of Human and Mouse DNA Probes to Determine the Chromosomal Contents of Cell Lines and Tumors

in Situ Hybridization Applicable to Abundantly Expressed mRNA Species

In Situ Hybridization of Frozen Sections Using 3 5S-Riboprobes

In Situ Detection of Human Papillomavirus DNA After Polymerase Chain Reaction Amplification

Accurate Quantitation of mRNA Species by Polymerase Chain Reaction and Solid-Phase Minisequencing