Contents of other Volumes ...................................... xi
Contributors ................................................ xxiii
Preface ....................................................... xiv
PART A. CELL AND TISSUE CULTURE: ASSOCIATED TECHNIQUES
Section 1. General Techniques
1. Setting up a Cell Culture Laboratory ......................... 5
ROBERT O'CONNOR AND LORRAINE O'DRISCOLL
2. General Procedures for Cell Culture ......................... 13
PAULA MELEADY AND ROBERT O'CONNOR
3. Counting Cells .............................................. 21
TRACY L. HOFFMAN
4. Cell Proliferation Assays: Improved Homogeneous Methods
Used to Measure the Number of Cells in Culture .............. 25
TERRY L. RISS AND RICHARD A. MORAVEC
5. Development of Serum-Free Media: Optimization of Nutrient
Composition and Delivery Format ............................. 33
DAVID W. JAYME AND DALE F. GRUBER
6. Cell Line Authentication .................................... 43
ROBERT J. HAY
7. Detection of Microbial and Viral Contaminants in Cell
Lines ....................................................... 49
ROBERT J. HAY AND PRANVERAIKONOMI
Section 2. Culture of Specific Cell Types: Stem Cells
8. Neural Crest Stem Cells ..................................... 69
MAURICE KLEBER AND LUKAS SOMMER
9. Postnatal Skeletal Stem Cells: Methods for Isolation and
Analysis of Bone Marrow Stromal Cells from Postnatal
Murine and Human Marrow ..................................... 79
SERGEI A. KUZNETSOV, MARA RIMINUCCI, PAMELA GEHRON
ROBEY, AND PAULO BIANCO
10.Establishment of Embryonic Stem Cell Lines from Adult
Somatic Cells by Nuclear Transfer ........................... 87
TERUHIKO WAKAYAMA
11.T-Cell Isolation and Propagation in vitro ................... 97
MADS HALD ANDERSEN AND PER THOR STRATEN
12.Generation of Human and Murine Dendritic Cells ............. 103
ANDREAS A.O. EGGERT, KERSTIN OTTO, ALEXANDER D.
MCLELLAN, PATRICK TERHEYDEN, CHRISTIAN LINDEN,
ECKHART KAMPGEN, AND JURGEN С. BECKER
Section 3. Culture of Specific Cell Types: Haemopoietic,
Mesenchymal, and Epithelial
13.Clonal Cultures in vitro for Haemopoietic Cells Using
Semisolid Agar Medium ...................................... 115
CHUNG LEUNG L.I., ANDREAS HUTTMANN, AND EUGENE
NGO-LUNG LAU
14.Human Skeletal Myocytes .................................... 121
ROBERT R. HENRY, THEODORE CIARALDI, AND SANDEEP
CHAUDHARY
15.Growing Madin-Darby Canine Kidney Cells for Studying
Epithelial Cell Biology .................................... 127
KAI SIMONS AND HIKKA VIRTA
16.Cultivation and Retroviral Infection of Human Epidermal
Keratinocytes .............................................. 133
FIONA M. WATT, SIMON BROAD, AND DAVID M. PROWSE
17.Three-Dimensional Cultures of Normal and Malignant
Human Breast Epithelial Cells to Achieve in vivo-like
Architecture and Function .................................. 139
CONNIE MYERS, HONG LIU, EVA LEE, AND MINA J. BISSELL
18.Primary Culture of Drosophila Embryo Cells ................. 151
PAUL M. SALVATERRA, IZUMI HAYASHI, MARTHA PEREZ-
MAGALLANES, AND KAZUO IKEDA
19. Laboratory Cultivation of Caenorhabditis elegans and
Other Free-Living Nematodes ............................... 157
IAN M. CALDICOTT, PAMELA L. LARSEN, AND DONALD
L. RIDDLE
Section 4. Differentiation and Reprogamming of Somatic
Cells
20.Induction of Differentiation and Cellular Manipulation
of Human Myeloid HL-60 Leukemia Cells ...................... 165
DAVID A. GLESNE AND ELIEZER HUBERMAN
21.Cultured PC12 Cells: A Model for Neuronal Function,
Differentiation, and Survival .............................. 171
KENNETH K. TENG, JAMES M. ANGELASTRO, MATTHEW E.
CUNNINGHAM, AND LLOYD A. GREENE
22.Differentiation of Pancreatic Cells into Hepatocytes ....... 177
DAVID TOSH
23.TERA2 and Its NTERA2 Subline: Pluripotent Human
Embryonal Carcinoma Cells .................................. 183
PETER W. ANDREWS
24.Embryonic Explants from Xenopus laevis as an Assay
System to Study Differentiation of Multipotent
Precursor Cells ............................................ 191
THOMAS HOLLEMANN, YONGLONG CHEN, MARION SOLTER,
MICHAEL KUHL, AND THOMAS PIELER
25.Electrofusion: Nuclear Reprogramming of Somatic Cells
by Cell Hybridization with Pluripotential Stem Cells ....... 199
MASAKO TADA AND TAKASHI TADA
26.Reprogramming Somatic Nuclei and Cells with Cell
Extracts ................................................... 207
ANNE-MARI HAKELIEN, HELGA B. LANDSVERK, THOMAS
KUNTZIGER, KRISTINE G. GAUSTAD, AND PHILIPPE COLLAS
Section 5. Immortalization
27.Immortalization of Primary Human Cells with Telomerase ..... 215
KWANGMOON LEE, ROBERT L. KORTUM, AND MICHEL M.
OUELLETTE
28.Preparation and Immortalization of Primary Murine Cells .... 223
NORMAN E. SHARPLESS
Section 6. Somatic Cell Hybrids
29.Viable Hybrids between Adherent Cells: Generation,
Yield Improvement, and Analysis ............................ 231
DORIS CASSIO
Section 7. Cell Separation Techniques
30.Separation and Expansion of Human T Cells .................. 239
AXL ALOIS NEURAUTER, TANJA AARVAK, LARS NORDERHAUG,
0YSTEIN AMELLEM, AND ANNE-MARIE RASMUSSEN
31.Separation of Cell Populations Synchronized in Cell
Cycle Phase by Centrifugal Elutriation ..................... 247
R. CURTIS BIRD
32.Polychromatic Flow Cytometry ............................... 257
STEPHEN P. PERFETTO, STEPHEN С DE ROSA, AND MARIO
ROEDERER
33.High-Speed Cell Sorting .................................... 269
SHERRIF F. IBRAHIM, TIMOTHY W. PETERSEN, JUNO СНОЕ,
AND GER VAN DEN ENGH
Section 8. Cell Cycle Analysis
34.Cell Cycle Analysis by Flow and Laser-Scanning Cytometry ... 279
ZBIGNIEW DARZYNKIEWICZ, PIOTR POZAROWSKI, AND GLORIA
JUAN
35.Detection of Cell Cycle Stages in situ in Growing Cell
Populations ................................................ 291
IRINA SOLOVEI, LOTHAR SCHERMELLEH, HEINER ALBIEZ,
AND THOMAS CREMER
36.In vivo DNA Replication Labeling ........................... 301
LOTHAR SCHERMELLEH
37.Live Cell DNA Labeling and Multiphoton/Confocal
Microscopy ................................................. 305
PAUL J. SMITH AND RACHEL J. ERRINGTON
Section 9. Cytotoxic and Cell Growth Assays
38.Cytotoxicity and Cell Growth Assays ........................ 315
GIUSEPPE S.A. LONGO-SORBELLO, GURAY SAYDAM,
DEBABRATA BANERJEE, AND JOSEPH R. BERTINO
39.Micronuclei and Comet Assay ................................ 325
ILONA WOLFF AND PEGGY MULLER
Section 10. Apoptosis
40.Methods in Apoptosis ....................................... 335
LORRAINE O'DRISCOLL, ROBERT O'CONNOR, AND MARTIN
CLYNES
Section 11. Assays of Cell Transformation, Tumorigenesis,
Invasion, and Wound Healing
41.Cellular Assays of Oncogene Transformation ................. 345
MICHELLE A. BOODEN, AYLIN S. ULKU, AND CHANNING
J. DER
42.Assays of Tumorigenicity in Nude Mice ...................... 353
ANNE-MARIE ENGEL AND MORTEN SCHOU
43.Transfilter Cell Invasion Assays ........................... 359
GARTH L. NICOLSON
44.Endothelial Cell Invasion Assay ............................ 363
NOONA AMBARTSUMIAN, CLAUS R. L. CHRISTENSEN, AND
EUGENE LUKANIDIN
45.Analysis of Tumor Cell Invasion in Organotypic Brain
Slices Using Confocal Laser-Scanning Microscopy ............ 367
TAKANORI OHNISHI AND HIRONOBU HARADA
46.Angiogenesis Assays ........................................ 373
YIHAI САО
47.Three-Dimensional, Quantitative, in vitro Assays of
Wound Healing Behavior ..................................... 379
DAVID. L. SHREIBER AND ROBERT T. TRANQUILLO
Section 12. Electrophysiological Methods
48.Patch Clamping ............................................. 395
BETH RYCROFT, FIONA С HALLIDAY, AND ALASDAIR J. GIBB
Section 13. Organ Cultures
49.Preparation of Organotypic Hippocampal Slice Cultures ...... 407
SCOTT M. THOMPSON AND SUSANNE E. MASON
50.Thyroid Tissue-Organotypic Culture Using a New Approach
for Overcoming the Disadvantages of Conventional Organ
Culture .................................................... 411
SHUJI TODA, AKIFUMI OOTANI, SHIGEHISA AOKI, AND
HAJIME SUGIHARA
PART B. VIRUSES
Section 14. Growth and Purification of Viruses
51.Growth of Semliki Forest Virus ............................. 419
MATHILDA SJOBERG AND HENRIK GAROFF
52.Design and Production of Human Immunodeficiency Virus-
Derived Vectors ............................................ 425
PATRICK SALMON AND DIDIER TRONO
53.Construction and Preparation of Human Adenovirus Vectors ... 435
MARY M. HITT, PHILLIP NG, AND FRANK L. GRAHAM
54.Production and Quality Control of High-Capacity
Adenoviral Vectors ......................................... 445
GUDRUN SCHIEDNER, FLORIAN KREPPEL, AND STEFAN
KOCHANEK
55.Novel Approaches for Production of Recombinant Adeno-
Associated Virus ........................................... 457
ANGELIQUE S. CAMP, SCOTT MCPHEE, AND R. JUDE SAMULSKI
PART С. ANTIBODIES
Section 15. Production and Purification of Antibodies
56.Production of Peptide Antibodies in Rabbits and
Purification of Immunoglobulin ............................. 467
GOTTFRIED PROESS
57.Preparation of Monoclonal Antibodies ....................... 475
PETER J. MACARDLE AND SHEREE BAILEY
58.Rapid Development of Monoclonal Antibodies Using
Repetitive Immunizations, Multiple Sites ................... 483
ERIC P. DIXON, STEPHEN SIMKINS, AND KATHERINE
E. KILPATRICK
59.Phage-Displayed Antibody Libraries ......................... 491
ANTONIETTA M. LILLO, KATHLEEN M. MCKENZIE, AND KIM
D. JANDA
60.Ribosome Display: In Vitro Selection of Protein-Protein
Interactions ............................................... 497
PATRICK AMSTUTZ, HANS KASPAR BINZ, CHRISTIAN ZAHND,
AND ANDREAS PLUCKTHUN
61.Epitope Mapping by Mass Spectrometry ....................... 511
CHRISTINE HAGER-BRAUN AND KENNETH B. TOMER
62.Mapping and Characterization of Protein Epitopes by
Using the SPOT Method ...................................... 519
CLAUDE GRANIER, SYLVIE VILLARD, AND DANIEL LAUNE
63.Determination of Antibody Specificity by Western
Blotting ................................................... 527
JULIO E. CELIS, JOSE M. A. MOREIRA, AND PAVEL GROMOV
64.Enzyme-Linked Immunoabsorbent Assay ........................ 533
STAFFAN PAULIE, PETER PERLMANN, AND HEDVIG PERLMANN
65.Radioiodination of Antibodies .............................. 539
STEPHEN J. MATHER
PART D. IMMUNOCYTOCHEMISTRY
Section 16. Immunofluoresence
66.Immunofluorescence Microscopy of Cultured Cells ............ 549
MARY OSBORN
67.Immunofluorescence Microscopy of the Cytoskeleton:
Combination with Green Flourescent Protein Tags ............ 557
JOHANNA PRAST, MARIO GIMONA, AND J. VICTOR SMALL
68.Immunocytochemistry of Frozen and of Paraffin Tissue
Sections ................................................... 563
MARY OSBORN AND SUSANNE BRANDFASS
PART E. APPENDIX
69.Representative Cultured Cell Lines and Their
Characteristics ............................................ 573
ROBERT J. HAY
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