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Introductionth to Cell and Tissue Culture - Jennie P.

Jennie P. Introductionth to Cell and Tissue Culture - Plenum Press, 2002.
ISBN 0-306-45859-4
Download (direct link): introductiontocellandtissueсulture2002.pdf
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Marcel Zocher, Kathy King, Glynis McCray, and the other past and present members of our laboratory. It is impossible to overestimate the contributions of these friends and colleagues who have, in the course of their work and studies in the Mather Laboratories over the years, added greatly to our knowledge and the fun of cell culture. Finally, we would like to thank Dr. Gordon Sato, who introduced us to the joy of cell culture and the infinite variety of interesting things to do with cells.
A note on the figures: The graphs and tables presented throughout the book are drawn from actual experimental data generated in the Mather Laboratories over the last 20 years. We have chosen those experiments that best illustrate the point being discussed in the text and have not necessarily provided all the experimental details for each figure.
We would also like to thank the following vendors for their help in discussions of their equipment and, where noted, in providing photographs or data for the figures and tables: James Quach, Instrument Services, Genentech, BRL Life Technologies, Corning Corporation, Falcon (Becton Dickinson), The Baker Company, Mike Alden of Coulter Electronics, E. Braun Biotech International, The Edge Scientific Instrument Co., Altair Gases, Sara Ferrer and Technical Instrument Company, and Brent Kolhede of Lab Equipment Company.
Page xi
Chapter 1: Introduction 1
The History of Tissue and Organ Culture 1
The Practice and Theory of Tissue Culture 3
Primary Culture 4
Established Cell Lines 6
The Physical and Chemical Environment 6
Complex versus Defined Culture Environments 7
Further Information 7
References 8
Chapter 2: Setting Up a Cell Culture Laboratory 9
Space Requirements 9
Equipment 11
The Teaching Laboratory 11
The Standard Tissue Culture Laboratory 13
The Optimal Tissue Culture Laboratory 19
Plasticware and Glassware 20
Maintaining the Laboratory 21
Daily Tasks 21
Weekly Tasks 23
Monthly Tasks 23
Chapter 3: The Physical Environment 25
Temperature 25
-pH 28
Osmolality 30
Page xii
CO2, Oxygen, and Other Gases 31
Surfaces and Cell Shape 32
Adherent versus Nonadherent Cells 33
PlasticjaDifferent Types for Different Purposes 34
Glass 35
-Cell Shape 36
Basement Membrane and Attachment Factors 36
Artificial Membranes 36
Stress 37
pH, Temperature, and Osmolality 39
Mechanical 39
Toxic Chemicals and Heavy Metals 39
Proteases 40
References 40
Chapter 4: Media 41
What Does the Medium Do? 41
Matching the Incubator Settings and the Medium 47
How to Select the Appropriate Medium 48
Media Preparation 51
Preparing Medium 52
Filter Sterilization 52
Serum Treatment 53
Testing Media and ComponentsjaQuality Control 54
Troubleshooting Medium Problems 55
Altering Commercial Media for Special Uses 56
Medium Optimization 57
References 61
Chapter 5: Standard Cell Culture Techniques 63
Subculturing 63
Subculturing Adherent Cells 64
Subculturing Suspension Cultures 65
Growth Curves and Measuring Cell Growth 66
Using the Hemacytometer or Electronic Particle Counter 67
Electronic Particle Counting 68
Generating a Growth Curve 70
Secondary Endpoint Assays for Proliferation 71
[3H]Thymidine Incorporation Assay for DNA Synthesis 72
High-Throughput Assays for Secondary Endpoints for Cell Number 73
Measuring Cell Viability 73
_Acridine Orange'CEthidium Bromide Viability Determination 74
Plating Efficiency 74
Conditioning Medium 76
Cloning 76
Cloning by Picking Colonies of Attached Cells 77
Cloning in Serum-Free Media 78
Cloning by Limiting Dilution 79
Page xiii
Freezing and Thawing Cells 80
Freezing 81
Temporary Freezing of Large Numbers of Clones 82
Thawing 83
Frozen Cell Storage 85
Record Keeping 85
Summary 86
References 87
Chapter 6: Looking at Cells 89
Just Look at the Dish 89
The Light Microscope Level 89
Phase Contrast 91
Hoffman or Nomarski Optics 95
Care and Handling of the Phase Contrast Microscope 96
Fluorescence Microscopy 97
Labeling Cells with a Fluorescent Viable Cell Dye 101
Cell Preparation, Fixation, and Antibody Binding 102
Bright Field 104
Dark Field 104
Adding the Third Dimension 106
Confocal Microscopy 106
Adding the Fourth Dimension 107
Real-Time Video 107
Time-Lapse Video 107
High-Speed Video 110
Looking More Closely 110
Scanning Electron Microscopy 112
Transmission Electron Microscopy 112
References 114
Chapter 7: Contamination: How to Avoid It, Recognize It, and Get Rid of It 117
Strings, Wigglies, and Pretty Balls of Fluff 117
Things You Cannot See Can Hurt You 119
Mycoplasma 119
Method for Fluorescent Detection of Mycoplasma 121
Virus 123
Cross-Culture Contamination 123
What Can You Do to Prevent Contamination? 125
What Can You Do to Get Rid of Contamination in Cultures? 126
References 127
Chapter 8: Serum-Free Culture 129
The Substitution of Defined Components for Serum 131
Preparation and Selection of Medium 135
Water 136
Page xiv
Preparing and Testing Hormones and Growth Factors 137
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