Description
Humana Flow Cytometry Principles And Applications 2007 Edition by Marion G. Macey
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. 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)2.3.1.3. 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 o