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This book provides a comprehensive introduction to the theory and applications of flow cytometry. Guidance is given on data interpretation, quality control procedures, pitfalls and problems, along with detailed protocols from leading authorities in flow cytometry. Flow Cytometry: Principles and Applications also presents the principles and potential of flow cytometry to assess functional aspects of cells.
1 Principles of flow cytometry. History and development of flow cytometry. Principles of flow cytometry. Fluorescence analysis. Light scatter and fluorescence detection. Acquisition. Amplification. Histograms. Coefficients of variation CV's. Spectral overlap and compensation. Safety aspects of lasers. Cell sorting. Commercial flow cytometers. 2 Cell preparation. Factors affecting the choice of prepation procedure: live versus fixed samples. Factors affecting cell preparation. Fixation: commonly used fixatives and their effects. Permeabilisation and the detection of intracellular components. Immunolabelling. Safety. 3 Fluorochromes and fluorescence. Interactions between light and matter. Light absorption leading to fluorescence. Mechanisms of fluorescent staining. Cellular autofluorescence. Fluorescence resonance energy transfer (FRET). Fluorochromes for labelling antibodies, proteins and ligands. Fluorochromes for labelling nucleic acids. Fluorescent probes for cell viability and apoptosis. Membrane integrity. Transmembrane potential. Apoptosis. Fluorescent probes for determining intracellular ion concentrations. Calcium ions. pH values. Fluorescent probes for phagocytosis and oxidative metabolism. Fluorochrome-labelled substrate analogues for measuring enzyme activity. Fluorescent dyes for measuring total protein. Fluorochromes combinations suitable for use in instruments equipped with a single laser emitting at 488 nm. Fluorochrome options when using instruments equipped with light sources additional to a 488 nm laser. 4 Quality control in flow cytometry. Internal Quality Assurance. Instrument Quality Control. Quality control issues and pitfalls. External Quality Assessment. Reagent selection. Definition of Positive Values. Absolute Count Enumeration. 5 Experimental design, data analysis and fluorescence quantitation. How many events should be acquired? The use of "thresholding" to enhance data acquisition rates. The choice of fluorochrome. The choice of monoclonal antibody clone (mAb) clone. The choice of "negative" control. The measurement of fluorescence intensity. 6 Apoptosis Detection By Flow Cytometry. Apoptotic Pathways and the Role of the Mitochondrion. Apoptosis and Necrosis. Viability and Necrosis. Apoptosis. Methodological Advantages and Disadvantages. Problems associated with DNA content assays. Problems associated with the TUNEL and phosphatydyl serine techniques. Problems associated with the detection of __m. Protocols. 7 DNA Analysis by Flow Cytometry. The Cell Cycle. Checkpoints. Numerical Chromosomal Aberrations. Dyes used to determine DNA content. Pulse Processing. Fixation and permeabilisation of cells for analysis. Protocols. 8 Immunological Studies of Human Cells. The identification of cells in human blood. Rare event analysis. Excluding non-specific labelled cells by using a dump channel. Using CFDA, SE. Intracellular labelling for cytokines and chemokines. 9 Calcium: Cytoplasmic, mitochondrial, endoplasmic reticulum and flux measurements. Procedure for tracking calcium movement within the cell. Indo-1 measurements of cytoplasmic calcium flux. Rhod-2 measurements of calcium flux to mitochondria. Changes in calcium within in a cell following receptor activation. Calcium flux assay procedure for Fluo-3. Further functional studies. Expression of functional antigens and receptors on the cell surface. Receptor signalling. Priming and activation. Prolonged responses to cytokines and/or hormones. Shape changes. Chemoattractant binding and rapid responses to chemotaxins/activators. Membrane potential and changes in ion permeability. Phagocytosis, endocytosis and oxidative burst. Alternative assays for phagocytosis. Nitric oxide release. Multiparameter techniques to assess function and phenotype. Adhesion molecules in cell-cell interactions. Analysis of cell-cell interactions. Platelet-platelet interactions. Flow cytometric analysis of platelet activation. Methods for the analysis of platelet adhesion and activation molecules. Platelet microparticle analysis. Platelet-leucocyte interactions. Leucocyte leuc
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)