Bacterial Binding, Phagocytosis, and Killing: Measurements Using Colony Forming Units -- Analysis of Human and Mouse Neutrophil Phagocytosis by Flow Cytometry -- Quantifying Phagocytosis by Immunofluorescence and Microscopy -- Methods for Quantitative Efferocytosis Assays -- Analysis of Efferocytic Receptor Dynamics and Synapse Formation in a Frustrated Efferocytosis Model -- Super-Resolution Spinning-Disk Confocal Microscopy Using Optical Photon Reassignment (SoRa) to Visualize the Actin Cytoskeleton in Macrophages -- Filamentous Bacteria as Targets to Study Phagocytosis -- The Derivation and Use of HoxB8-Driven Conditionally Immortalized Macrophages -- Quantitative Immunofluorescence to Study Phagosome Maturation and Resolution -- Approaches to Measuring Reductive and Oxidative Events in Phagosomes -- Measuring Phagosomal pH by Fluorescence Microscopy -- Multiplexed Phagosomal Assays for the Detection and Quantification of Bidirectional Exchange Between the Phagolysosomal Lumen and Extracellular Space -- Quantitative Spatio-Temporal Analysis of Phagosome Maturation in Live Cells -- Measurement of Salmonella enterica Internalization and Vacuole Lysis in Epithelial Cells -- Microscopy-Based Tracking and Quantification Methods to Study Phagosome Resolution -- Isolation of Polystyrene Bead-Induced Phagosomes for Western Blotting -- Biochemically Reconstituted Fusion of Phagosomes with Endosomes and Lysosomes -- An In Vitro System to Analyze Generation and Degradation of Phagosomal Phosphatidylinositol Phosphates -- Examining Phagosomal Pattern Recognition Receptor-Dependent Signaling and Antigen MHC-II Presentation from Phagosomes in Dendritic Cells -- Examining the Kinetics of Phagocytosis-Coupled Inflammasome Activation in Murine Bone Marrow-Derived Dendritic Cells -- Analysis of LC3-Associated Phagocytosis and Antigen Presentation in Macrophages and B Cells -- Visualizing Phagocytic Cargo In Vivo from Engulfment to Resolution in Caenorhabditis elegans -- Assessing the Phagosome Proteome by Quantitative Mass Spectrometry -- Using Ion Substitution and Fluid Indicators to Monitor Macropinosome Dynamics in Live Cells.

This volume explores updated and entirely new experimental approaches used to investigate phagocytosis and phagosome maturation. In order to aid in the study of engulfment, maturation, resolution, and pathogen manipulation of phagocytes, the book features methodology to quantify uptake and maturation specific to certain phagocytes, particles, or pathogens, while other chapters offer methods that can be applied generically across the field. Methods are presented to study phagocytosis and phagosome maturation in vivo, in cellulo, and through in vitro analyses. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step and readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and up-to-date, Phagocytosis and Phagosomes: Methods and Protocols, Second Edition serves as a vital resource for both experts in the field as well as for investigators delving into phagocytosis and phagosome maturation for the first time.

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