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This volume aims to cover all major methodological aspects of research into purinergic signaling and to provide a foundation for studying them at molecular, biochemical, pharmacological, and physiological levels. Chapters guide readers through current knock-out and knock-in mouse models, in silico modeling, knock down purinoceptor expression, bioluminiscence resonance energy transfer, enzyme-based biosensors, recording P2X receptor electrophysiology, controlling P2X receptors by optogenetics, inflammasome activation, leukocyte migration, and cell adhesion. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions 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, Purinergic Signaling: Methods and Protocols will provide asound basis for molecular, cellular, and physiological research into purinergic signaling in health and disease and will spark interest in this fascinating signaling process among researchers in many different and unrelated disciplines.
List of contents
Introduction to Purinergic Signaling.- Knock-out and Knock-in Mouse Models to Study Purinergic Signaling.- Agonists and Antagonists for Purinergic Receptors.- Homology Modelling of P2X Receptors.- Using RNA Interference for Purinoceptor Knock Down in vivo.- Developmental Expression of Ectonucleotidase and Purinergic Receptors Detection by Whole Mount in situ hybridization in Xenopus Embryos.- Histochemical Approach for Simultaneous Detection of Ecto-nucleotidase and Alkaline Phosphatase Activities in Tissues.- Flow Cytometry of Membrane Purinoreceptors.- Studying Purinoceptor Cell-surface Expression by Protein Biotinylation.- Multimeric Ionotropic Purinoceptor Detection by Protein Cross-linking.- Multimeric Purinoceptor Detection by Bioluminiscence Resonance Energy Transfer.- Application of Fluorescent Purinoceptor Antagonists for Bioluminescence Resonance Energy Transfer Assays and Fluorescent Microscopy.- Detection of Extracellular ATP in the Tumor Microenvironment, using the pmeLUC Biosensor.- Using Amperometric, Enzyme-Based Biosensors for Performing Longitudinal Measurements of Extracellular adenosine 5-triphosphate in the Mouse.- Fluorescent Labeling and Quantification of Vesicular ATP Release using Live Cell Imaging.- Using FRET-based Fluorescent Sensors to Monitor Cytosolic and Membrane-proximal Extracellular ATP levels .- ATP Measurement in Cerebrospinal Fluid Using a Microplate Reader.- P2X Electrophysiology and Surface Trafficking in Xenopus oocytes.- Heterologous Expression and Patch-clamp Recording of P2X Receptors in HEK293 Cells.- Recording P2X Receptors Using Whole Cell Patch Clamp from Native Monocytes and Macrophages.- Automated Planar Patch-clamp Recording of P2X Receptors.- Controlling Engineered P2X Receptors with Light.- Intracellular Calcium Recording after Purinoceptor Activation Using a Video-microscopy Equipment.- Assays to Measure Purinoceptor Pore Dilation.- Detection of Inflammasome Activation by P2X7 Purinoceptor Activation by Determining ASC Oligomerization.- Measuring Leukocyte Migration to Nucleotides.- Assessment of Cell Adhesion after Purinoceptor Activation.
