Purinergic Signaling, 1st ed. 2020
Methods and Protocols
Methods in Molecular Biology Series, Vol. 2041

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 a sound 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.

1. Introduction to Purinergic Signaling

Geoffrey Burnstock

2. Knock-out and Knock-in Mouse Models to Study Purinergic Signaling

Robin M. H. Rumney and Dariusz C. Górecki

3. Agonists and Antagonists for Purinergic Receptors

Christa E. Müller and Vigneshwaran Namasivayam

4. Homology Modelling of P2X Receptors

Anastasios Stavrou, Sudad Dayl, and Ralf Schmid

5. Using RNA Interference for Purinoceptor Knock Down in vivo 

Rebeca Padrão Amorim, Iscia Teresinha Lopes Cendes, and Maria Jose da Silva Fernandes

6. Developmental Expression of Ectonucleotidase and Purinergic Receptors Detection by Whole Mount in situ hybridization in Xenopus Embryos

Camille Blanchard and Karine Massé

7. Histochemical Approach for Simultaneous Detection of Ecto-nucleotidase and Alkaline Phosphatase Activities in Tissues

Karolina Losenkova, Marius Paul, Heikki Irjala, Sirpa Jalkanen, and Gennady G. Yegutkin

8. Flow Cytometry of Membrane Purinoreceptors

Nicole Schwarz, Marten Junge, Friedrich Haag, and Friedrich Koch-Nolte

9. Studying Purinoceptor Cell-surface Expression by Protein Biotinylation

10. Multimeric Ionotropic Purinoceptor Detection by Protein Cross-linking

Vincent Compan and François Rassendren

11. Multimeric Purinoceptor Detection by Bioluminiscence Resonance Energy Transfer

Vincent Compan and François Rassendren

12. Application of Fluorescent Purinoceptor Antagonists for Bioluminescence Resonance Energy Transfer Assays and Fluorescent Microscopy

Mark Soave, Joëlle Goulding, Robert Markus, Stephen J Hill, and Leigh A Stoddart

13. Detection of Extracellular ATP in the Tumor Microenvironment, using the pmeLUC Biosensor

Elena De Marchi, Elisa Orioli, Anna Pegoraro, Elena Adinolfi, and Francesco Di Virgilio

14. Using Amperometric, Enzyme-Based Biosensors for Performing Longitudinal Measurements of Extracellular adenosine 5-triphosphate in the Mouse

Edward Beamer and Tobias Engel

15. Fluorescent Labeling and Quantification of Vesicular ATP Release using Live Cell Imaging

Kirstan A. Vessey, Tracy Ho, Andrew I. Jobling, Yao A. Wang, and Erica L. Fletcher

17. ATP Measurement in Cerebrospinal Fluid Using a Microplate Reader

Laura de Diego-García, Álvaro Sebastián-Serrano, Carolina Bianchi, Caterina Di Lauro, and Miguel Díaz-Hernández

18. P2X Electrophysiology and Surface Trafficking in Xenopus oocytes

Eléonore Bertin, Audrey Martinez, and Eric Boué-Grabot

19. Heterologous Expression and Patch-clamp Recording of P2X Receptors in HEK293 Cells

Lin-Hua Jiang and Sébastien Roger

20. Recording P2X Receptors Using Whole Cell Patch Clamp from Native Monocytes and Macrophages

Leanne Stokes

21. Automated Planar Patch-clamp Recording of P2X Receptors

Carol J. Milligan and Lin-Hua Jiang

22. Controlling Engineered P2X Receptors with Light

23. Intracellular Calcium Recording after Purinoceptor Activation Using a Video-microscopy Equipment

Maria Teresa Miras-Portugal, Felipe Ortega, Javier Gualix, Raquel Perez-Sen, Esmerilda G. Delicado, and Rosa Gomez-Villafuertes

24. Assays to Measure Purinoceptor Pore Dilation

Ben J. Gu, Pavan Avula and James S. Wiley

25. Detection of Inflammasome Activation by P2X7 Purinoceptor Activation by Determining ASC Oligomerization

26. Measuring Leukocyte Migration to Nucleotides

Taylor J. Moon and Michael R. Elliott

27. Assessment of Cell Adhesion after Purinoceptor Activation

Juan J. Martínez-García and Pablo Pelegrín

Includes cutting-edge methods and protocols

Provides step-by-step detail essential for reproducible results

Contains key notes and implementation advice from the experts