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Pratical cell analysis by Dimitri Pappas 2010
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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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