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This detailed volume explores the NADPH oxidase family of enzymes in human physiology and genetic disease, in which early discoveries represent prime examples of the finest translational "from bed to bench and back" studies. Methods are included for testing assembly and function of multicomponent oxidase complexes and for analyzing reactive oxygen species (ROS) generation in different systems by various means, while addressing pitfalls of ROS probes currently being used, as well as protocols on NADPH oxidase regulation and their function in cells. Written in the highly successful Methods in Molecular Biology series format, chapters include introduction to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and cutting-edge, NADPH Oxidases: Methods and Protocols will aid researchers working with the NOX/DUOX family in continuing and expanding upon their vital research.
List of contents
Intersecting Stories of the Phagocyte NADPH Oxidase and Chronic Granulomatous Disease.- Mammalian NADPH Oxidases.- Enhanced Immunoaffinity Purification of Human Neutrophil Flavocytochrome B for Structure Determination by Electron Microscopy.- Purification and Characterization of DUOX Peroxidase Homology Domains (PHDs).- A Close-Up View of the Impact of Arachidonic Acid on the Phagocyte NADPH Oxidase.- NOX5 Cell-Free Assay for the High-Throughput Screening of Small Molecules.- Spectroscopy of NOX Protein Family Members.- Soluble Regulatory Proteins for Activation of NOX Family NADPH Oxidases.- Insights into the NOX NADPH Oxidases Using Heterologous Whole Cell Assays.- The X-CGD PLB-985 Cell Model for NOX2 Structure-Function Analysis.- Functional Characterization of DUOX Enzymes in Reconstituted Cell Models.- Guidelines for the Detection of NADPH Oxidases by Immunoblot and RT-qPCR.- Methods for Detection of NOX-Derived Superoxide Radical Anion and Hydrogen Peroxide in Cells.- HPLC-BasedMonitoring of Oxidation of Hydroethidine for Detection of NADPH Oxidase-Derived Superoxide Radical Anion.- Visualization of Intracellular Hydrogen Peroxide with the Genetically-Encoded Fluorescent Probe HyPer in NIH-3T3 Cells.- Imaging Intracellular H2O2 with the Genetically-Encoded PerFRET and OxyFRET Probes.- Quantitative Imaging of Endogenous and Exogenous H2O2 Gradients in Live Zebrafish Larvae.- Kinetic Analysis of Phagosomal ROS Generation.- Imaging Intestinal ROS in Homeostatic Conditions Using L-012.- Hydro-Cy3-Mediated Detection of Reactive Oxygen Species In Vitro and In Vivo.- Phosphorylation of Gp91phox/NOX2 in Human Neutrophils.- The Molecular Regulation and Functional Roles of NOX5.- Using Synthetic Peptides for Exploring Protein-Protein Interactions in the Assembly of the NADPH Oxidase Complex.- Rational Design and Delivery of NOX-Inhibitory Peptides.- High-Throughput Screening of NOX Inhibitors.- Protein-Protein Interaction Assay to Analyze NOX4/p22phox Heterodimerization.- Isolation of Redox Active Endosomes (Redoxosomes) and Assessment of NOX Activity.- Model Systems to Investigate NOX-Dependent Cell Migration and Invasiveness.- NADPH Oxidases and Aging Models of Lung Fibrosis.- Proteomic Methods to Evaluate NOX-Mediated Redox Signaling.- Neutrophil Extracellular Traps.- Chronic Granulomatous Disease.- Diagnostic Testing for Chronic Granulomatous Disease.- Gastrointestinal Complications in Chronic Granulomatous Disease.- Ex Vivo Models of Chronic Granulomatous Disease.- Gene Editing in Chronic Granulomatous Disease.- DUOX Defects and Their Roles in Congenital Hypothyroidism.- NAPDH Oxidases in Inflammatory Bowel Disease.
Summary
This detailed volume explores the NADPH oxidase family of enzymes in human physiology and genetic disease, in which early discoveries represent prime examples of the finest translational “from bed to bench and back” studies. Methods are included for testing assembly and function of multicomponent oxidase complexes and for analyzing reactive oxygen species (ROS) generation in different systems by various means, while addressing pitfalls of ROS probes currently being used, as well as protocols on NADPH oxidase regulation and their function in cells. Written in the highly successful Methods in Molecular Biology series format, chapters include introduction to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and cutting-edge, NADPH Oxidases: Methods and Protocols will aid researchers working with the NOX/DUOX family in continuing and expanding upon their vital research.
