Within this practical guide:
- The procedures and protocols for cell separation, handling cells on a microscope and for using cells in microfluidic devices are presented.
- Elements of cell culture are taken and combined with the practical advice necessary to maintain a cell lab and to handle cells properly during an analysis
- The main chapters deal with the fundamentals and applied aspects of each technique, with one complete chapter focusing on statistical considerations of analyzing cells
- Many diagram-based protocols for some of the more common cell processes are included
- Chapter summaries and extensive tables are included so that key information can be looked up easily in the lab setting
Much like a good manual or cookbook this book is a useful, practical guide and a handy reference for all students, researchers and practitioners involved in cellular analysis.
Table of Contents
Chapter 1: Getting Starting (and Getting the Cells)1.1 Introduction
1.2 The Driving Need
1.3 Primary and Cultured Cells
1.4 Choosing a Cultured Cell
1.5 Choosing Primary Cells
1.6 Easily Obtainable Primary Cells
1.7 Primary Cells from Tissues
1.8 Purifying Primary Cells
1.9 How Long do Primary Cells Remain Primary?
1.10 Obtaining Primary Cells from a Commercial Source
1.11 Bacteria and Yeast
1.12 Practical Aspects of Cell Culture
1.13 Safety Aspects of Primary and Transformed Cell Lines
1.14 Transfection of Primary and Transformed Cell Lines
1.15 Conclusion
References
Chapter 2: The Cell Culture Laboratory (Tools of the Trade)
2.1 Introduction
2.2 Issues Concerning a Cell Laboratory
2.3 Setting up a Cell Culture Laboratory
2.4 Cell Line Storage
2.5 Personal Protective Equipment
2.6 Cell and Sample Handling
2.7 Common Analytical Instrumentation for Cell Culture
2.8 Considerations when Setting up a Cell Culture Laboratory
2.9 Establishing and Regulating A Culture Facility
2.10 Conclusion
References
Chapter 3. Maintaining Cultures
3.1 Introduction
3.2 Medium
3.3 The Use of Medium in Analysis, and Alternatives
3.4 Culturing Cells
Protocol 3.1: Sub-Culture of Adherent Cells
3.5 Growing Cells in Three Dimensions
3.6 Sterility and Contamination of Culture
3.7 Storage of Cell Samples and Cell Lines
Protocol 3.2: Cryopreservation of Mammalian Cells
Protocol 3.3: Retrieval of Cells from Liquid Nitrogen Storage
3.8 Conclusion
References
Chapter 4. Microscopy of Cells
4.1 Introduction
4.2 Microscope Types
4.3 Culturing Cells for Microscopy
4.4 Signals, Background, and Artifacts in Optical Microscopy
4.5 Staining Cells for Fluorescence Microscopy
4.6 Multiple Labels
4.7 Viability and Two-Photon Microscopy
4.8 Spatial Resolution in Optical Microscopy
4.9 Image Saturation and Intensity
4.10 Atomic Force and Environmental Scanning Electron Microscopy
4.11 Conclusion
References
Chapter 5: Separating Cells
5.1 Introduction
5.2 The Cell Sample
5.3 Label-Free (Intrinsic) Separations
5.4 Immunomagnetic Sorting
5.5 Cell Affinity Chromatography
5.6 Affinity Chemistry Considerations in CAC and MACS Separations
Protocol 5.1: Screening of Antibody Clones
5.7 Elution in Cell Affinity Chromatography
5.8 Nonspecific Binding in Cell Separations
5.9 Separation of Rare Cells
5.10 Fluorescence Activate Cell Sorting
5.11 Sorting Parameters
5.12 Other Separation Techniques and Considerations
5.13 Conclusion
References
Chapter 6. Flow Cytometry: Cell Analysis in the Fast Lane
6.1 Introduction
6.2 The Cell Sample
6.3 Flow Cytometer Function
6.4 Obtaining or Finding a Flow Cytometer
6.5 Using Flow Cytometers
6.6 Setting up a Flow Cytometer for Multi-Color Staining
6.7 Analyzing Flow Cytometry Data
6.8 Example Flow Cytometry Assays
6.9 No-Flow Cytometry
6.10 Conclusion
References
Chapter 7: Analyzing Cells with Microfluidic Devices
7.1 Introduction
7.2 Advantages of Microfluidics
7.3 Considerations of Microfluidics and Cells
7.4 Obtaining Microfluidic Cell Devices
7.5 Microfluidic Flow Cytometry
7.6 Cell Separations
7.7 Analysis of Cell Products
7.8 Cell Culture
Protocol 7.1: Low-shear Cell Culture Chip
7.9 Conclusion
References
Chapter 8: Statistical Considerations
8.1 Introduction
8.2 Types of Error
8.3 Figures of Merit in Statistical Analysis of Cells
8.4 Limits of Detection and Quantitation (of Cell)
8.5 Methods to Improve Cell Statistics
8.6 Comparing Analytical Values
8.7 Rejecting Data: Proceed With Caution
8.8 Conclusion
References
Chapter 9 Protocols, Probes, and Standards
9.1 Introduction
9.2 Cell Transfection and Immortalization (Chapter 1)
Protocol 9.1: Transfecting Cells with Polyamine Reagents
Protocol 9.2: Stable Transfection using Polyamine Delivery
Protocol 9.3: Transfection Using Electroporation
Optimizing Electroporation Parameters
Protocol 9.4: Cell Immortalization using hTERT Transfection
9.3 Calculating Relative Centrifugal Force (RCF) and Centrifuge Rotor Speed (Chapter 2)
9.4 Fluorescence Methods (Chapter 4 & 6)
Protocol 9.4: Apoptosis Detection Using Fluorophore-Conjugated Annexin-V and a Viability Dye
Protocol 9.5: Apoptosis Detection Using Fluorogenic Caspase Probes
9.5 Surface Modifications for Cell Analysis (Chapter 5 & 7)
Protocol 9.6: Covalent Linkage of Proteins (non-Antibody) to Glass by Microcontact Imprinting
Protocol 9.7: Covalent Linkage of Antibodies to Glass
Protocol 9.8: Noncovalent Attachment of Antibodies to Glass #1
Protocol 9.9: Noncovalent Attachment of Antibodies to Glass or PDMS #2
Protocol 9.10: Blocking Endogenous Biotin
9.6 Flow Cytometry and Cell Separations (Chapter 5 & 6)
Protocol 9.11: Cell Cycle Measurements by Flow Cytometry
Protocol 9.12: Antigen Density Measurements in Flow Cytometry
Protocol 9.13: Antigen Density Measurements using Fluorescence Correlation Spectroscopy.
Protocol 9.14: Cell Proliferation using Anti-CD71 staining (Chapter 4 & 6).
9.7 Fluorescent Labels and Fluorogenic Probes (Chapter 4, 6 & 7)
References
