Flow Cytometry
Principles and Applications
(Sprache: Englisch)
Flow cytometry forms an integral part of both basic biological research and clinical diagnosis in pathology. This straightforward new volume provides a clear, easy-to-read, and practical manual for both clinicians and non-clinicians at all levels of their...
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Klappentext zu „Flow Cytometry “
Flow cytometry forms an integral part of both basic biological research and clinical diagnosis in pathology. This straightforward new volume provides a clear, easy-to-read, and practical manual for both clinicians and non-clinicians at all levels of their careers.
The chapter topics range from basic principles to more advanced subjects, such as apoptosis and cell sorting. The book charts the history, development and basic principles of flow cytometry.
Inhaltsverzeichnis zu „Flow Cytometry “
1. Principles of flow cytometry- Marion G Macey
1.1 History and development of flow cytometry
1.2 Principles of flow cytometry
1.3 Fluorescence analysis
1.4 Light scatter and fluorescence detection
1.5 Acquisition
1.6 Amplification
1.7 Histograms
1.8 Coefficients of variation CV's
1.9 Spectral overlap and compensation
1.10 Safety aspects of lasers
1.11 Cell sorting
1.12 Commercial flow cytometers
1.13 References
2. Cell preparation
- Desmond A. McCarthy
2.1. Introduction
2.2. Factors affecting the choice of prepation procedure: live versus fixed samples
2.3. Factors affecting cell preparation
2.3.1. Processing blood and bone marrow samples
2.3.1.1. Live whole blood procedures
2.3.1.2. Leucocyte isolation techniques (Dextran sedimentation, Density gradient centrifugation, Immunoselection, Erythrocyte lysis)
2.3.1.3. Lysed whole blood procedures
2.3.2. Preparation of cell suspensions from organs, tissues and cell cultures
2.4 Fixation: commonly used fixatives and their effects
2.5. Permeabilisation and the detection of intracellular components
2.6. Immunolabelling
2.6.1. Antibodies
2.6.2. Antibody-antigen interactions
2.6.3. Antibody titration
2.6.4. Sensitivity of detection and the measurement of cell surface antigens
2.6.5. Direct and indirect immunostaining
2.6.6. Determining absolute cell counts
2.7. Safety
2.8. References
3. Fluorochromes and fluorescence
- Desmond A. McCarthy
3.1. Introduction
3.2. Interactions between light and matter
3.3. Light absorption leading to fluorescence
3.4. Mechanisms of fluorescent staining
3.5. Cellular autofluorescence
3.6. Fluorescence resonance energy transfer (FRET)
3.7. Fluorochromes for labelling antibodies, proteins and ligands
3.7.1. Fluorescein and other green fluorescent fluorochromes
3.7.2. Phycobiliproteins (phycoerythrin, allophycocyanin and CryptoFluor" dyes) and peridinin-chlorophyll a complex (PerCP)
3.7.3 Tandem dyes
3.7.4. Quantum dots
3.7.5. Conjugation of
... mehr
fluorochromes to antibodies, proteins or small ligands
3.7.6. Indirect immunolabelling with fluorochromes conjugated to protein A or G, avidin or streptavidin
3.8. Fluorochromes for labelling nucleic acids
3.9. Fluorescent probes for cell viability and apoptosis
3.9.1 Membrane integrity
3.9.2. Transmembrane potential
3.9.3 Apoptosis
3.10. Fluorescent probes for determining intracellular ion concentrations
3.10.1. Calcium ions
3.10.2. pH values
3.11. Fluorescent probes for phagocytosis and oxidative metabolism
3.11.1. Phagocytosis
3.11.2. Oxidative metabolism
3.12. Fluorochrome-labelled substrate analogues for measuring enzyme activity
3.13. Fluorescent dyes for measuring total protein
3.14. Fluorochromes combinations suitable for use in instruments equipped with a single laser emitting at 488 nm
3.15. Fluorochrome options when using instruments equipped with light sources additional to a 488 nm laser
3.16. References.
4. Quality control in flow cytometry
- David Barnett and John T Reilly
4.1 Introduction
4.2 Internal Quality Assurance
4.3 Instrument Quality Control
4.3 Quality control issues and pitfalls
4.4 External Quality Assessment
4.5 Reagent selection
4.6 Definition of Positive Values
4.7 Absolute Count Enumeration
4.8 Conclusion
4.9 References.
5. Experimental design, data analysis and fluorescence quantitation.
- Mark Lowdell
5.1 Introduction
5.2 How many events should be acquired?
5.3 The use of "thresholding" to enhance data acquisition rates.
5.4 The choice of fluorochrome.
5.6 The choice of monoclonal antibody clone (mAb) clone.
5.7 The choice of "negative" control.
5.8 The measurement of fluorescence intensity.
5.9 References.
6. Apoptosis Detection By Flow Cytometry
- Paul Allen and Derek Davies
6.1 Introduction.
6.2 Apoptotic Pathways and the Role of the Mitochondrion.
6.3 Apoptosis and Necrosis.
6.4 Viability and Necrosis.
6.5 Apoptosis.
6.6 Methodological Advantages and Disadvantages.
6.7 Problems associated with DNA content assays.
6.8 Problems associated with the TUNEL and phosphatydyl serine techniques.
6.9 Problems associated with the detection of __m .
6.10 Protocols.
6.11 References.
7. DNA Analysis by Flow Cytometry
- Derek Davies and Paul Allen
7.1 Introduction
7.2 The Cell Cycle
7.3 Checkpoints
7.4 Numerical Chromosomal Aberrations
7.5 Dyes used to determine DNA content.
7.6 Pulse Processing.
7.7 Fixation and permeabilisation of cells for analysis
7.8 Protocols
7.9 References.
8. Immunological Studies of Human Cells
- Ulrika Johansson
8.1 The identification of cells in human blood.
8.2. Rare event analysis.
8.2.1 The statistics of rare events: How many cells to acquire?
8.2.2 Other considerations for small populations.
8.3. Excluding non-specific labelled cells by using a dump channel.
8.3.1 Dead cells, red cells and debris.
8.3.2. Sample carryover.
8.4.2. Using CFDA, SE
8.4.1. The dyes available for the analysis of cell proliferation.
8.4.2. Using CFDA, SE
8.4.3. Labelling cells with CFDA, SE.
8.4.4. CFDA, SE labelling protocol.
8.4.5. Controls for proliferation and for antigen labelling.
8.4.6. Analysis of CSFE labelled cells.
8.4.7. How to calculate proliferation and graphically display data.
8.4.8. Measuring cell subset expansion in mixed cell cultures.
8.5. Intracellular labelling for cytokines and chemokines
8.5.1. Blocking protein transport
8.5.2. Monensin and Brefeldin A: wanted and unwanted effects.
8.5.3. Cell activation, control populations and time of harvest.
8.5.4. Labeling procedure for intracellular cytokines.
8.6 References.
9. Calcium: Cytoplasmic, mitochondrial, endoplasmic reticulum and flux measurements.
- Gary Warnes and Marion Macey.
9.1 Introduction.
9.2 Procedure for tracking calcium movement within the cell.
9.3 Indo-1 measurements of cytoplasmic calcium flux.
9.4 Rhod-2 measurements of calcium flux to mitochondria.
9.5 Changes in calcium within in a cell following receptor activation.
9.6 Calcium flux assay procedure for Fluo-3
9.7 References.
10. Further functional studies
- Marion G Macey.
10.1 Introduction
10.2 Expression of functional antigens and receptors on the cell surface
10.3 Receptor signalling
10.4 Priming and activation
10.5 Prolonged responses to cytokines and/or hormones
10.7 Shape changes
10.6 Chemoattractant binding and rapid responses to chemotaxins/activators
10.8 Membrane potential and changes in ion permeability
10.9 Phagocytosis, endocytosis and oxidative burst
10.10 Alternative assays for phagocytosis
10.11 Nitric oxide release
10.12 Multiparameter techniques to assess function and phenotype
10.13 Adhesion molecules in cell-cell interactions
10.14 Analysis of cell-cell interactions
10.15 Platelet-platelet interactions
10.16 Flow cytometric analysis of platelet activation
10.17 Methods for the analysis of platelet adhesion and activation molecules
10.18 Platelet microparticle analysis
10.19 Platelet-leucocyte interactions
10.20 Leucocyte leucocyte interactions
10.21 Leucocyte endothelial cell interactions
10.22 References
11. Cell Sorting by Flow Cytometry
- Derek Davies
11.1 Introduction
11.2. Applications
11.3 Principles of particle sorting
11.3.1 Electrostatic sorting
11.3.2 Mechanical and other forms of sorting
11.4 Practicalities of cell sorting
11.4.1 Sample preparation
11.4.2 Preparation from suspension cells
11.4.3 Preparation from adherent cells
11.4.4 Preparation from solid tissue
11.4.5 Sort set up
11.4.6 Tips and Troubleshooting
11.5 Health and Safety Considerations
11.6 References
- Appendix A
- Useful internet sites
3.7.6. Indirect immunolabelling with fluorochromes conjugated to protein A or G, avidin or streptavidin
3.8. Fluorochromes for labelling nucleic acids
3.9. Fluorescent probes for cell viability and apoptosis
3.9.1 Membrane integrity
3.9.2. Transmembrane potential
3.9.3 Apoptosis
3.10. Fluorescent probes for determining intracellular ion concentrations
3.10.1. Calcium ions
3.10.2. pH values
3.11. Fluorescent probes for phagocytosis and oxidative metabolism
3.11.1. Phagocytosis
3.11.2. Oxidative metabolism
3.12. Fluorochrome-labelled substrate analogues for measuring enzyme activity
3.13. Fluorescent dyes for measuring total protein
3.14. Fluorochromes combinations suitable for use in instruments equipped with a single laser emitting at 488 nm
3.15. Fluorochrome options when using instruments equipped with light sources additional to a 488 nm laser
3.16. References.
4. Quality control in flow cytometry
- David Barnett and John T Reilly
4.1 Introduction
4.2 Internal Quality Assurance
4.3 Instrument Quality Control
4.3 Quality control issues and pitfalls
4.4 External Quality Assessment
4.5 Reagent selection
4.6 Definition of Positive Values
4.7 Absolute Count Enumeration
4.8 Conclusion
4.9 References.
5. Experimental design, data analysis and fluorescence quantitation.
- Mark Lowdell
5.1 Introduction
5.2 How many events should be acquired?
5.3 The use of "thresholding" to enhance data acquisition rates.
5.4 The choice of fluorochrome.
5.6 The choice of monoclonal antibody clone (mAb) clone.
5.7 The choice of "negative" control.
5.8 The measurement of fluorescence intensity.
5.9 References.
6. Apoptosis Detection By Flow Cytometry
- Paul Allen and Derek Davies
6.1 Introduction.
6.2 Apoptotic Pathways and the Role of the Mitochondrion.
6.3 Apoptosis and Necrosis.
6.4 Viability and Necrosis.
6.5 Apoptosis.
6.6 Methodological Advantages and Disadvantages.
6.7 Problems associated with DNA content assays.
6.8 Problems associated with the TUNEL and phosphatydyl serine techniques.
6.9 Problems associated with the detection of __m .
6.10 Protocols.
6.11 References.
7. DNA Analysis by Flow Cytometry
- Derek Davies and Paul Allen
7.1 Introduction
7.2 The Cell Cycle
7.3 Checkpoints
7.4 Numerical Chromosomal Aberrations
7.5 Dyes used to determine DNA content.
7.6 Pulse Processing.
7.7 Fixation and permeabilisation of cells for analysis
7.8 Protocols
7.9 References.
8. Immunological Studies of Human Cells
- Ulrika Johansson
8.1 The identification of cells in human blood.
8.2. Rare event analysis.
8.2.1 The statistics of rare events: How many cells to acquire?
8.2.2 Other considerations for small populations.
8.3. Excluding non-specific labelled cells by using a dump channel.
8.3.1 Dead cells, red cells and debris.
8.3.2. Sample carryover.
8.4.2. Using CFDA, SE
8.4.1. The dyes available for the analysis of cell proliferation.
8.4.2. Using CFDA, SE
8.4.3. Labelling cells with CFDA, SE.
8.4.4. CFDA, SE labelling protocol.
8.4.5. Controls for proliferation and for antigen labelling.
8.4.6. Analysis of CSFE labelled cells.
8.4.7. How to calculate proliferation and graphically display data.
8.4.8. Measuring cell subset expansion in mixed cell cultures.
8.5. Intracellular labelling for cytokines and chemokines
8.5.1. Blocking protein transport
8.5.2. Monensin and Brefeldin A: wanted and unwanted effects.
8.5.3. Cell activation, control populations and time of harvest.
8.5.4. Labeling procedure for intracellular cytokines.
8.6 References.
9. Calcium: Cytoplasmic, mitochondrial, endoplasmic reticulum and flux measurements.
- Gary Warnes and Marion Macey.
9.1 Introduction.
9.2 Procedure for tracking calcium movement within the cell.
9.3 Indo-1 measurements of cytoplasmic calcium flux.
9.4 Rhod-2 measurements of calcium flux to mitochondria.
9.5 Changes in calcium within in a cell following receptor activation.
9.6 Calcium flux assay procedure for Fluo-3
9.7 References.
10. Further functional studies
- Marion G Macey.
10.1 Introduction
10.2 Expression of functional antigens and receptors on the cell surface
10.3 Receptor signalling
10.4 Priming and activation
10.5 Prolonged responses to cytokines and/or hormones
10.7 Shape changes
10.6 Chemoattractant binding and rapid responses to chemotaxins/activators
10.8 Membrane potential and changes in ion permeability
10.9 Phagocytosis, endocytosis and oxidative burst
10.10 Alternative assays for phagocytosis
10.11 Nitric oxide release
10.12 Multiparameter techniques to assess function and phenotype
10.13 Adhesion molecules in cell-cell interactions
10.14 Analysis of cell-cell interactions
10.15 Platelet-platelet interactions
10.16 Flow cytometric analysis of platelet activation
10.17 Methods for the analysis of platelet adhesion and activation molecules
10.18 Platelet microparticle analysis
10.19 Platelet-leucocyte interactions
10.20 Leucocyte leucocyte interactions
10.21 Leucocyte endothelial cell interactions
10.22 References
11. Cell Sorting by Flow Cytometry
- Derek Davies
11.1 Introduction
11.2. Applications
11.3 Principles of particle sorting
11.3.1 Electrostatic sorting
11.3.2 Mechanical and other forms of sorting
11.4 Practicalities of cell sorting
11.4.1 Sample preparation
11.4.2 Preparation from suspension cells
11.4.3 Preparation from adherent cells
11.4.4 Preparation from solid tissue
11.4.5 Sort set up
11.4.6 Tips and Troubleshooting
11.5 Health and Safety Considerations
11.6 References
- Appendix A
- Useful internet sites
... weniger
Bibliographische Angaben
- 2007, 294 Seiten, Maße: 15,9 x 23,7 cm, Gebunden, Englisch
- Herausgegeben:Macey, Marion G.
- Herausgegeben: Marion G. Macey
- Verlag: Humana Press
- ISBN-10: 1588296911
- ISBN-13: 9781588296917
Sprache:
Englisch
Rezension zu „Flow Cytometry “
From the reviews:"This book 'Flow Cytometry: Principles and Applications' focuses on flow cytometry as being an integral part of both basic biological research and clinical diagnosis in pathology. This volume provides a clear and practical manual especially for non-clinicians working in the clinical or experimental laboratory. ... immunologists and haematologists in the field of research, as well as biological researchers working with both human and animal models, will appreciate this book." (Jan Philippé, Acta Clinica Belgica, Vol. 63 (2), 2008)
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