Practical Cell Analysis

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ISBN: 9780470741559 | 0470741554
Cover: Hardcover
Copyright: 4/5/2010

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Practical Cell Analysis serves as a guide for researchers interested in adding cell analyses to their repertoire. This book covers the basics of setting up a cell culture-capable laboratory, as well as sources and types of cells. In addition, the book also discusses the procedures for cell separation, handling cells on a microscope, and using cells in microfluidic devices. Cell viability, probe toxicity, apoptosis, cell storage, and other practical stumbling blocks are also discussed. Each chapter features useful tables such as common cell fluorescence probes and separation methods/protocols.

Preface	p. xiii
Acknowledgments	p. xix
Getting Started (and Getting the Cells)	p. 1
Introduction	p. 1
The Driving Need	p. 2
Primary and Cultured Cells	p. 3
Choosing a Cultured Cell	p. 6
Choosing Primary Cells	p. 11
Easily Obtainable Primary Cells	p. 14
Primary Cells from Tissues	p. 16
Purifying Primary Cells	p. 21
How Long do Primary Cells Remain Primary?	p. 24
Obtaining Primary Cells from a Commercial Source	p. 25
Bacteria and Yeast	p. 26
Practical Aspects of Cell Culture	p. 27
Safety Aspects of Primary and Transformed Cell Lines	p. 29
Transfection of Primary and Transformed Cell Lines	p. 30
Conclusion	p. 32
References	p. 32
The Cell-Culture Laboratory (Tools of the Trade)	p. 35
Introduction	p. 35
Issues Concerning a Cell Laboratory	p. 36
Setting up a Cell Culture Laboratory	p. 44
Cell Line Storage	p. 48
Personal Protective Equipment	p. 51
Cell and Sample Handling	p. 52
Common Analytical Instrumentation for Cell Culture	p. 53
Considerations when Setting up a Cell-Culture Laboratory	p. 57
Establishing and Regulating a Culture Facility	p. 61
Conclusion	p. 63
References	p. 63
Maintaining Cultures	p. 65
Introduction	p. 65
Medium	p. 66
The Use of Medium in Analysis, and Alternatives	p. 71
Culturing Cells	p. 73
Sub-Culture of Adherent Cells	p. 75
Growing Cells in Three Dimensions	p. 77
Sterility and Contamination of Culture	p. 79
Storage of Cell Samples and Cell Lines	p. 80
Cryopreservation of Mammalian Cells	p. 83
Retrieval of Cells from Liquid-Nitrogen Storage	p. 84
Conclusion	p. 86
References	p. 86
Microscopy of Cells	p. 89
Introduction	p. 89
Microscope Types	p. 90
Culturing Cells for Microscopy	p. 95
Signals, Background, and Artifacts in Optical Microscopy	p. 101
Staining Cells for Fluorescence Microscopy	p. 104
Fixation of Cells for Immunochemical Staining	p. 111
Multiple Labels	p. 113
Viability and Two-Photon Microscopy	p. 116
Spatial Resolution in Optical Microscopy	p. 117
Image Saturation and Intensity	p. 119
Atomic Force and Environmental Scanning Electron Microscopy	p. 120
Conclusion	p. 121
References	p. 122
Separating Cells	p. 125
Introduction	p. 125
The Cell Sample	p. 126
Label-Free (Intrinsic) Separations	p. 131
Immunomagnetic Sorting	p. 134
Cell-Affinity Chromatography	p. 137
Affinity Chemistry Considerations in CAC and MACS Separations	p. 142
Screening of Antibody Clones	p. 145
Elution in Cell-Affinity Chromatography	p. 147
Nonspecific Binding in Cell Separations	p. 148
Separation of Rare Cells	p. 151
Fluorescence-Activated Cell Sorting	p. 152
Sorting Parameters	p. 156
Other Separation Techniques and Considerations	p. 156
Conclusion	p. 160
References	p. 161
Flow Cytometry: Cell Analysis in the Fast Lane	p. 165
Introduction	p. 165
The Cell Sample	p. 167
Flow Cytometer Function	p. 169
Obtaining or Finding a Flow Cytometer	p. 177
Using Flow Cytometers	p. 178
Setting up a Flow Cytometer for Multi-Color Staining	p. 181
Analyzing Flow Cytometry Data	p. 185
Example Flow-Cytometry Assays	p. 189
No-Flow Cytometry	p. 191
Conclusion	p. 192
References	p. 192
Analyzing Cells with Microfluidic Devices	p. 195
Introduction	p. 195
Advantages of Microfluidics	p. 196
Considerations of Microfluidics and Cells	p. 198
Obtaining Microfluidic Cell Devices	p. 202
Microfluidic Flow Cytometry	p. 209
Cell Separations	p. 213
Analysis of Cell Products	p. 215
Cell Culture	p. 219
Low-Shear Cell-Culture Chip	p. 222
Conclusion	p. 225
References	p. 225
Statistical Considerations	p. 229
Introduction	p. 229
Types of Error	p. 230
Figures of Merit in Statistical Analysis of Cells	p. 236
Limits of Detection and Quantitation (of Cell)	p. 240
Methods to Improve Cell Statistics	p. 242
Comparing Analytical Values	p. 243
Rejecting Data: Proceed With Caution	p. 245
Conclusion	p. 245
References	p. 246
Protocols, Probes, and Standards	p. 247
Introduction	p. 247
Cell Transfection and Immortalization (Chapter 1)	p. 247
Transfecting Cells with Polyamine Reagents	p. 248
Stable Transfection using Polyamine Delivery	p. 249
Transfection Using Electroporation	p. 251
Cell Immortalization Using hTERT Transfection	p. 255
Calculating Relative Centrifugal Force (RCF) and Centrifuge Rotor Speed (Chapter 2)	p. 256
Fluorescence Methods (Chapters 4 and 6)	p. 256
Apoptosis Detection Using Fluorophore-Conjugated Annexin-V and a Viability Dye	p. 257
Apoptosis Detection Using Fluorogenic Caspase Probes	p. 263
Surface Modifications for Cell Analysis (Chapters 5 and 7)	p. 266
Covalent Linkage of Proteins (Nonantibody) to Glass by Microcontact Imprinting	p. 266
Covalent Linkage of Antibodies to Glass	p. 269
Noncovalent Attachment of Antibodies to Glass #1	p. 271
Noncovalent Attachment of Antibodies to Glass or PDMS #2	p. 272
Blocking Endogenous Biotin	p. 273
Flow Cytometry and Cell Separations (Chapters 5 and 6)	p. 274
Cell Cycle Measurements by Flow Cytometry	p. 274
Antigen Density Measurements in Flow Cytometry	p. 275
Antigen Density Measurements Using Fluorescence Correlation Spectroscopy	p. 279
Cell Proliferation Using Anti-CD71 Staining (Chapters 4 and 6)	p. 281
Fluorescent Labels and Fluorogenic Probes (Chapters 4-7)	p. 283
References	p. 284
Index	p. 287
Table of Contents provided by Ingram. All Rights Reserved.

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Practical Cell Analysis

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