Practical Cell Analysis

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.

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