ISBN-10:
0470741554
ISBN-13:
9780470741559
Pub. Date:
04/19/2010
Publisher:
Wiley
Practical Cell Analysis / Edition 1

Practical Cell Analysis / Edition 1

by Dimitri Pappas

Hardcover

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Product Details

ISBN-13: 9780470741559
Publisher: Wiley
Publication date: 04/19/2010
Pages: 314
Product dimensions: 6.20(w) x 9.00(h) x 0.90(d)

About the Author

Dimitri Pappas is Assistant Professor in Analytical Chemistry at Texas Tech University. He obtained his B.S., University of Florida, 1998 followed by his Ph.D., University of Florida, 2002. He worked as a Research Scientist at Wyle Laboratories, NASA/Johnson Space Center, 2002-05 before taking up his post at Texas Tech. His key areas of research are: Analytical Fluorescence and Raman Spectroscopy; Cellular Analysis; Flow Cytometry; Optics
With the focus on the interface between cell science and analytical chemistry. Dr. Pappas is currently developing novel analytical methods to study intact cells, cell digests, and tissue aggregates using spectroscopic and immunochemical techniques. The majority of his group’s research is geared toward clinical analysis as well as medical testing in remote areas.

Table of Contents

Preface xiii

Acknowledgments xix

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 48

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 122

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 222

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 273

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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