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[资源] Pratical cell analysis by Dimitri Pappas 2010

Pratical cell analysis by Dimitri Pappas 2010

Contents
1 Getting Started (and Getting the Cells) 1
1.1 Introduction 1
1.2 The Driving Need 2
1.3 Primary and Cultured Cells 3
1.4 Choosing a Cultured Cell 6
1.5 Choosing Primary Cells 11
1.6 Easily Obtainable Primary Cells 14
1.7 Primary Cells from Tissues 16
1.8 Purifying Primary Cells 21
1.9 How Long do Primary Cells Remain Primary? 24
1.10 Obtaining Primary Cells from a Commercial Source 25
1.11 Bacteria and Yeast 26
1.12 Practical Aspects of Cell Culture 27
1.13 Safety Aspects of Primary and Transformed
Cell Lines 29
1.14 Transfection of Primary and Transformed
Cell Lines 30
1.15 Conclusion 32
References 32

2 The Cell-Culture Laboratory (Tools of the Trade) 35
2.1 Introduction 35
2.2 Issues Concerning a Cell Laboratory 36
2.3 Setting up a Cell Culture Laboratory 44
2.4 Cell Line Storage
2.5 Personal Protective Equipment 51
2.6 Cell and Sample Handling 52
2.7 Common Analytical Instrumentation for Cell Culture 53
2.8 Considerations when Setting up a Cell-Culture
Laboratory 57
2.9 Establishing and Regulating a Culture Facility 61
2.10 Conclusion 63
References 63

3 Maintaining Cultures 65
3.1 Introduction 65
3.2 Medium 66
3.3 The Use of Medium in Analysis, and Alternatives 71
3.4 Culturing Cells 73
Protocol 3.1: Sub-Culture of Adherent Cells 75
3.5 Growing Cells in Three Dimensions 77
3.6 Sterility and Contamination of Culture 79
3.7 Storage of Cell Samples and Cell Lines 80
Protocol 3.2: Cryopreservation of Mammalian Cells 83
Protocol 3.3: Retrieval of Cells from Liquid-Nitrogen
Storage 84
3.8 Conclusion 86
References 86

4 Microscopy of Cells 89
4.1 Introduction 89
4.2 Microscope Types 90
4.3 Culturing Cells for Microscopy 95
4.4 Signals, Background, and Artifacts in Optical
Microscopy 101
4.5 Staining Cells for Fluorescence Microscopy 104
Protocol 4.1 Fixation of Cells for Immunochemical
Staining 111
4.6 Multiple Labels 113
4.7 Viability and Two-Photon Microscopy 116
4.8 Spatial Resolution in Optical Microscopy 117
4.9 Image Saturation and Intensity 119
4.10 Atomic Force and Environmental Scanning
Electron Microscopy 120
4.11 Conclusion 121
References

5 Separating Cells 125
5.1 Introduction 125
5.2 The Cell Sample 126
5.3 Label-Free (Intrinsic) Separations 131
5.4 Immunomagnetic Sorting 134
5.5 Cell-Affinity Chromatography 137
5.6 Affinity Chemistry Considerations in CAC and
MACS Separations 142
Protocol 5.1: Screening of Antibody Clones 145
5.7 Elution in Cell-Affinity Chromatography 147
5.8 Nonspecific Binding in Cell Separations 148
5.9 Separation of Rare Cells 151
5.10 Fluorescence-Activated Cell Sorting 152
5.11 Sorting Parameters 156
5.12 Other Separation Techniques and Considerations 156
5.13 Conclusion 160
References 161

6 Flow Cytometry: Cell Analysis in the Fast Lane 165
6.1 Introduction 165
6.2 The Cell Sample 167
6.3 Flow Cytometer Function 169
6.4 Obtaining or Finding a Flow Cytometer 177
6.5 Using Flow Cytometers 178
6.6 Setting up a Flow Cytometer for Multi-Color Staining 181
6.7 Analyzing Flow Cytometry Data 185
6.8 Example Flow-Cytometry Assays 189
6.9 No-Flow Cytometry 191
6.10 Conclusion 192
References 192

7 Analyzing Cells with Microfluidic Devices 195
7.1 Introduction 195
7.2 Advantages of Microfluidics 196
7.3 Considerations of Microfluidics and Cells 198
7.4 Obtaining Microfluidic Cell Devices 202
7.5 Microfluidic Flow Cytometry 209
7.6 Cell Separations 213
7.7 Analysis of Cell Products 215
7.8 Cell Culture 219
Protocol 7.1: Low-Shear Cell-Culture Chip
7.9 Conclusion 225
References 225

8 Statistical Considerations 229
8.1 Introduction 229
8.2 Types of Error 230
8.3 Figures of Merit in Statistical Analysis of Cells 236
8.4 Limits of Detection and Quantitation (of Cell) 240
8.5 Methods to Improve Cell Statistics 242
8.6 Comparing Analytical Values 243
8.7 Rejecting Data: Proceed With Caution 245
8.8 Conclusion 245
References 246

9 Protocols, Probes, and Standards 247
9.1 Introduction 247
9.2 Cell Transfection and Immortalization (Chapter 1) 247
Protocol 9.1: Transfecting Cells with Polyamine
Reagents 248
Protocol 9.2: Stable Transfection using Polyamine
Delivery 249
Protocol 9.3: Transfection Using Electroporation 251
Protocol 9.4: Cell Immortalization Using
hTERT Transfection 255
9.3 Calculating Relative Centrifugal Force (RCF) and
Centrifuge Rotor Speed (Chapter 2) 256
9.4 Fluorescence Methods (Chapters 4 and 6) 256
Protocol 9.4: Apoptosis Detection Using
Fluorophore-Conjugated Annexin-V and a
Viability Dye 257
Protocol 9.5: Apoptosis Detection Using
Fluorogenic Caspase Probes 263
9.5 Surface Modifications for Cell Analysis
(Chapters 5 and 7) 266
Protocol 9.6: Covalent Linkage of Proteins
(Nonantibody) to Glass by Microcontact Imprinting 266
Protocol 9.7: Covalent Linkage of Antibodies to Glass 269
Protocol 9.8: Noncovalent Attachment of Antibodies
to Glass #1 271
Protocol 9.9: Noncovalent Attachment of Antibodies
to Glass or PDMS #2 272
Protocol 9.10: Blocking Endogenous Biotin
9.6 Flow Cytometry and Cell Separations
(Chapters 5 and 6) 274
Protocol 9.11: Cell Cycle Measurements by Flow
Cytometry 274
Protocol 9.12: Antigen Density Measurements
in Flow Cytometry 275
Protocol 9.13: Antigen Density Measurements Using
Fluorescence Correlation Spectroscopy 279
Protocol 9.14: Cell Proliferation Using Anti-CD71
Staining (Chapters 4 and 6) 281
9.7 Fluorescent Labels and Fluorogenic Probes
(Chapters 4–7) 283
References 284
Index 287

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