Vol. 1. Culture and Biochemical Analysis of Cells
SECTION 1 Cell Culture and Analysis
1. Mammalian Cell Culture
2. Growth and Manipulation of Cells in Culture
3. Extracellular Matrix Components as Substrata in Cell and Tissue Culture
4. Isolation and Culture of Fibroblasts
5. Culture of Epithelial Cells
6. Isolation of Human Capillary Endothelial Cells
7. Isolation and Transformation of Lymphocytes
8. Culture and In Vitro Differentiation of Mouse Embryonic Stem Cells
9. Isolation and Culture of Primary Neural Cells
10. Isolation and Culture of Chick Embryo Myoblasts
11. Isolation and Culture of Neonatal Rat Cardimyocytes
12. Isolation and Culture of Adult Rat Ventricular Myocytes
13. Fusion of Somatic Cells
14. Cell Synchronization
15. Apoptosis Assays
16. Flow Cytometry
17. Culture and Manipulation of Hypotrichous Ciliated Protozoa
18. Culture and Manipulation of Tetrahymena
19. Culture and Genetic Analysis of Chlamydomonas
20. Culture and Analysis of Dictyostelium discoideum
21. Growth and Transformation of Saccharomyces cerevisiae
22. Growth and Transformation of Schizosaccharomyces pombe
23. Culture of Marine Invertebrate Embryos

SECTION 2 Metabolic Labeling and Protein Modification
24. Steady-State Labeling of HeLa Cells with 32P Phosphate
25. In Vitro Nuclear Run-on Analysis of mRNA Transcription
26. Metabolic Radiolabeling of Proteins
27. Metabolic Labeling of Cells with 32P for Studies of Protein hosphorylation
28. Study of Protein Phosphorylation Using Permeabilized Cells and Organelles
29. Protein Kinase Assays Using Exogenous Substrates
30. Kinetics for Kinases: Simple Enzyme Kinetics Applied to Two-substrate Enzymes
31. Simple Preparations of Protein Phosphatases
32. Methylation and Prenylation of Proteins
33. Protein Glycosylation
SECTION 3 Subcellular Fractionation
34. Subcellular Fractionation
35. Plasma Membrane Isolation Using the Cationic Colloidal Silica Isolation Technique
36. Isolation of Desmosomes
37. Subcellular Fractionation of Rough Microsomes
38. Purification of Ribosomes, Ribosomal Subunits, and Polysomes
39. Golgi Apparatus Isolation and Characterization
40. Isolation of Peroxisomes (Microbodies) from Mammalian Tissues
41. Isolation of Mitochondria from Cells and Tissues
42. Isolation of Intact Chloroplasts
43. Preparation of Nuclei from Tissue and Suspension Cultures
44. The Nuclear Matrix: Preparation for Microscopy and Biochemical Analysis
45. Permeabilized Cell Assay for Nuclear Protein Import
46. Analysis of Nucleocytoplasmic Shuttling Using Transient Interspecies Heterokaryons
47. Preparation and Characterization of the Nuclear Lamina and Nuclear Laminaenriched Fractions
48. Isolation of Nucleoli
49. Chromosome Isolation for Biochemical and Morphological Analysis
50. Purification and Southern Blotting Analysis of DNA
51. Purification and Northern Blot Analysis of Total RNA from Cells and Tissues
52. Spread Preparation of Xenopus Germinal Vesicle Contents
53. Purfication of Myosin and Actin from Muscle and Nonmuscle Cells
54. Isolation of Microtubules, MAPs, and Microtubule-dependent Motor Proteins
55. Isolation and Purification of Intermediate Filaments

SECTION 4 Protein Identification and Analysis
56. Determination of Protein Concentration
57. One-Dimensional Gel Electrophoresis of Proteins
58. Two-Dimensional Gel Electrophoresis of Proteins
59. Detection of Proteins by Staining
60. Detection of Proteins by Autoradiography, Fluorography, and Phosphor Imaging
61. One-Dimensional and Two-Dimensional Peptide Mapping
62. Peptide Mapping and Peptide Purification by RP-HPLC
63. Sequencing of Proteins and Peptides

SECTION 5 Protein Expression and Interactions
64. Producing Proteins in a T7 Expression System
65. Expression and Purification of Proteins Using GST Gene Fusion Vectors
66. Baculovirus Expression: Generation of Recombinant Baculovirus DNA in E. coli Using a Baculovirus Shuttle Vector
67. Mammalian Expression Vectors
68. An Autoregulatory, Tetracycline-controlled System for Inducible
Gene Expression in Mammalian Cells
69. Two-hybrid System/Interaction Trap

SECTION 6 Antibodies as Tools in Cell Biology
70. Overview of Antibody Purification and Labeling
71. Epitope Tagging
72. Immunoprecipitation
73. Immunoblotting and Immunoblot Affinity Purification
74. Human Autoantibodies and Characterization of Their Target Antigens

VOL 2. Light Microscopy and Cell Structure
SECTION 7 Observation of Live Cells and Cellular Dynamics
75. Observation of Live Cells in the Light Microscope
76. Monitoring Molecular Dynamics In Vivo Using Fluorescence Techniques
77. Phagokinetic Track Assay of Cell Locomotion in Tissue Culture
78. Heterologous Expression of the Green Fluorescent Protein
79. Fluorescence Photobleaching Techniques
80. Imaging and Measurement of Intracellular Free Calcium In Living Cells
81. Construction of Optical Tweezers

SECTION 8 Preparation of Macromolecules and Introduction into Cells
82. Fluorescence Labeling of Antibodies and DNA Probes
83. Quantitative Microinjection of Living Cells
84. Injection of Xenopus Oocytes and Embryos
85. Direct Injection of DNA into Rodent Muscle
86. Transfection of DNA into Mammalian Cells
87. Delivery of DNA into Mammalian Cells Using Cationic Liposomes
88. Electroporation and Electrofusion
89. Delivery of Antisense Oligonucleotides
90. Use and Application of Adenovirus Expression Vectors
91. Replication-defective Herpesvirus Amplicon Vectors and Their Use for Gene Transfer
92. Transduction of Genes Using Retroviral Vectors
93. Lineage Analysis Using Retroviral Vectors

SECTION 9 Light and Epifluorescence Microscopy
94. Light Microscopy
95. Video Microscopy and Image Enhancement

SECTION 10 Confocal Microscopy, Multiphoton Microscopy, and Deconvolution
96. Confocal Microscopy and Deconvolution Techniques
97. Multiphoton Excitation Fluorescence Microscopy

Vol. 3. Subcellular Localization of Genes and Their Products
SECTION 11 Visualization of Organelles, Proteins, and Gene Expression
98. Preparation of Cells and Tissues for Fluorescence Microscopy
99. Detection of xxx-Galactosidase and Alkaline Phosphatase Activities in Tissues
100. Immunolocalization of Proteins Using Enzyme Detection
101. Nonimmunological Fluorescent Labeling of Cellular Structures
102. Introduction to Immunofluorescence Microscopy
103. Immunostaining of Microtubules, Microtubule-associated Proteins, and Intermediate Filaments
104. Immunofluorescence Localization of Actin
105. Immunofluorescence Localization of Nuclear Proteins
106. Immunofluorescence Methods for Saccharomyces cerevisiae
107. Immunofluorescence Methods for Drosophila Tissues
108. Immunofluorescence Methods for C. elegans
109. Analyzing DNA Replication: Nonisotopic Labeling
110. Analyzing RNA Synthesis: Nonisotopic Labeling

SECTION 12 In Situ Hybridization
111. Fluorescence In Situ Hybridization to DNA
112. Comparative Genomic Hybridization
113. Chromosome Analysis by Spectral Karyotyping
114. In Situ Hybridization of DNA and Nuclear RNA in Tissue Squashes Using (3)H-labeled Probes
115. Whole-Mount Fluorescence In Situ Hybridization to Drosophila Chromosomal DNA
116. In Situ Hybridization to RNA
117. Whole-Mount In Situ Detection of RNAs in Vertebrate Embryos and Isolated Organs
118. Whole-Mount In Situ Detection of RNAs in Xenopus Embryos
119. In Situ Polymerase Chain Reaction

SECTION 13 Electron Microscopy
120. Image Production Using Transmission Electron Microscopy
121. Preparative Methods for Transmission Electron Microscopy
122. Image Production Using Scanning Electron Microscopy
123. Preparative Methods for Scanning Electron Microscopy
124. Scanning Transmission Electron Microscopy
125. Electron Microscopy of DNA and DNA-binding Proteins
126. Snapshot Blotting of Nucleic Acids and Nucleoprotein Complexes
127. Cytochemical