Flow Cytometry: Principles and Applications by Marion G. MaceyFlow Cytometry: Principles and Applications by Marion G. Macey

Flow Cytometry: Principles and Applications

EditorMarion G. Macey

Paperback | November 5, 2010

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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.

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Title:Flow Cytometry: Principles and ApplicationsFormat:PaperbackDimensions:304 pages, 9.02 × 5.98 × 0.07 inPublished:November 5, 2010Publisher:Humana PressLanguage:English

The following ISBNs are associated with this title:

ISBN - 10:1617377201

ISBN - 13:9781617377204

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Table of Contents

1 Principles of flow cytometryMarion G Macey1.1 History and development of flow cytometry1.2 Principles of flow cytometry1.3 Fluorescence analysis1.4 Light scatter and fluorescence detection1.5 Acquisition1.6 Amplification1.7 Histograms1.8 Coefficients of variation CV's1.9 Spectral overlap and compensation1.10 Safety aspects of lasers1.11 Cell sorting1.12 Commercial flow cytometers1.13 References2 Cell preparationDesmond A. McCarthy 2.1. Introduction2.2. Factors affecting the choice of prepation procedure: live versus fixed samples2.3. Factors affecting cell preparation2.3.1. Processing blood and bone marrow samples2.3.1.1. Live whole blood procedures2.3.1.2. Leucocyte isolation techniques (Dextran sedimentation, Density gradient centrifugation, Immunoselection, Erythrocyte lysis) Lysed whole blood procedures2.3.2. Preparation of cell suspensions from organs, tissues and cell cultures2.4 Fixation: commonly used fixatives and their effects2.5. Permeabilisation and the detection of intracellular components2.6. Immunolabelling2.6.1. Antibodies2.6.2. Antibody-antigen interactions2.6.3. Antibody titration2.6.4. Sensitivity of detection and the measurement of cell surface antigens2.6.5. Direct and indirect immunostaining2.6.6. Determining absolute cell counts2.7. Safety2.8. References3 Fluorochromes and fluorescenceDesmond A. McCarthy 3.1. Introduction3.2. Interactions between light and matter3.3. Light absorption leading to fluorescence3.4. Mechanisms of fluorescent staining3.5. Cellular autofluorescence3.6. Fluorescence resonance energy transfer (FRET)3.7. Fluorochromes for labelling antibodies, proteins and ligands3.7.1. Fluorescein and other green fluorescent fluorochromes3.7.2. Phycobiliproteins (phycoerythrin, allophycocyanin and CryptoFluor" dyes) and peridinin-chlorophyll a complex (PerCP)3.7.3 Tandem dyes3.7.4. Quantum dots3.7.5. Conjugation of fluorochromes to antibodies, proteins or small ligands3.7.6. Indirect immunolabelling with fluorochromes conjugated to protein A or G, avidin or streptavidin3.8. Fluorochromes for labelling nucleic acids3.9. Fluorescent probes for cell viability and apoptosis3.9.1 Membrane integrity3.9.2. Transmembrane potential3.9.3 Apoptosis3.10. Fluorescent probes for determining intracellular ion concentrations 3.10.1. Calcium ions3.10.2. pH values3.11. Fluorescent probes for phagocytosis and oxidative metabolism3.11.1. Phagocytosis3.11.2. Oxidative metabolism3.12. Fluorochrome-labelled substrate analogues for measuring enzyme activity3.13. Fluorescent dyes for measuring total protein3.14. Fluorochromes combinations suitable for use in instruments equipped with a single laser emitting at 488 nm3.15. Fluorochrome options when using instruments equipped with light sources additional to a 488 nm laser3.16. References.4 Quality control in flow cytometryDavid Barnett and John T Reilly4.1 Introduction4.2 Internal Quality Assurance4.3 Instrument Quality Control4.3 Quality control issues and pitfalls4.4 External Quality Assessment4.5 Reagent selection4.6 Definition of Positive Values4.7 Absolute Count Enumeration4.8 Conclusion4.9 References.5 Experimental design, data analysis and fluorescence quantitation. Mark Lowdell5.1 Introduction5.2 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 CytometryPaul Allen and Derek Davies6.1 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 CytometryDerek Davies and Paul Allen7.1 Introduction7.2 The Cell Cycle7.3 Checkpoints7.4 Numerical Chromosomal Aberrations 7.5 Dyes used to determine DNA content.7.6 Pulse Processing.7.7 Fixation and permeabilisation of cells for analysis7.8 Protocols7.9 References.8 Immunological Studies of Human CellsUlrika Johansson8.1 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, SE8.4.1. The dyes available for the analysis of cell proliferation.8.4.2. Using CFDA, SE8.4.3. 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 chemokines8.5.1. 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-39.7 References.10 Further functional studiesMarion G Macey.10.1 Introduction 10.2 Expression of functional antigens and receptors on the cell surface10.3 Receptor signalling10.4 Priming and activation10.5 Prolonged responses to cytokines and/or hormones10.7 Shape changes10.6 Chemoattractant binding and rapid responses to chemotaxins/activators10.8 Membrane potential and changes in ion permeability10.9 Phagocytosis, endocytosis and oxidative burst10.10 Alternative assays for phagocytosis10.11 Nitric oxide release10.12 Multiparameter techniques to assess function and phenotype10.13 Adhesion molecules in cell-cell interactions10.14 Analysis of cell-cell interactions10.15 Platelet-platelet interactions10.16 Flow cytometric analysis of platelet activation 10.17 Methods for the analysis of platelet adhesion and activation molecules10.18 Platelet microparticle analysis10.19 Platelet-leucocyte interactions10.20 Leucocyte leucocyte interactions10.21 Leucocyte endothelial cell interactions10.22 References11 Cell Sorting by Flow CytometryDerek Davies11.1 Introduction11.2. Applications11.3 Principles of particle sorting 11.3.1 Electrostatic sorting11.3.2 Mechanical and other forms of sorting11.4 Practicalities of cell sorting11.4.1 Sample preparation11.4.2 Preparation from suspension cells11.4.3 Preparation from adherent cells11.4.4 Preparation from solid tissue11.4.5 Sort set up11.4.6 Tips and Troubleshooting11.5 Health and Safety Considerations11.6 ReferencesAppendix AUseful internet sites.

Editorial Reviews

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)