Bioluminescence

This second edition details the newest and most innovative techniques in this rapidly expanding field. It provides methods and assays that can be adapted and used in most specialties of biomedical and biotechnological fields.

Part I I wasraised in a redbrick Baltimore row housewhere summer was marked by the ti- honored ritual of firefly-chasing a backyard tradition that has endured the gene- tions.Amidtheexcitement,myfatheroftentoldthestoryofhow,whenhewasachild, researchers at the Johns Hopkins University had appealed for the systematic capture of live fireflies en masse. Science had engaged the Baltimore youth in an entrepreneurial quest to jar as many lightning bugs as the dwindling light of dusk would permit. The very next morning, each 100-count glass jar of glowing crawling insects could be exchanged at the University for exactly one crisp dollar bill. Unrecognized at the time by my father, his joyous endeavors had contributed in a profoundwaytotheadvancedmolecularbiologicaltechniquesthatserveasthebasisfor thistextbook.In1947,WilliamMcElroyusedextractsfromthoseveryfirefliestodefine the fundamental reaction underlying the mystical phenomenon of luminescence, and published ''The energy source for bioluminescence in an isolated system'' in the Proceedings of the National Academy of Sciences. Inthedecadessincethatsummer,thestudyandapplicationofbioluminescencehave allowed us to leverage the enduring power of nature's elegance. We have painstakingly harnessedapowerfultoolthatenablesustoseekadeeperunderstandingofthecomplex mechanisms underpinning so many vital biologic systems. This second edition of Methods in Molecular Biology's Bioluminescence: Methods and Protocols serves as a re- ableandutilitariancompilationofthenewestandmostinnovativetechniquesthathave emerged in this rapidly expanding and progressively diverse field. We are indebted to the authors for their thoughtful contributions, inspired by their rigorous dedication to the science of bioluminescence, humbled by theunyielding supportofourcolleagues,andgratefulfortheopportunityprovidedusbyJohnandJan Walker.

Provides an easily accessible reference volume for a wide range of bioluminescence assays and applications written by leading researchers in the field Comprehensive guide to developing, validating, running, and analyzing bioluminescence assays Develops detection and imaging methods Describes the use of a wide variety of bioluminescence tools: various luciferases, reporter gene systems including PIN-G reporters, light emitting bacteria, smart probes, BRET, quantums dots, transgenic animals, etc. Presents protocols with enough details to be followed and adapted by scientific teams with no previous experience in bioluminescence Contains numerous notes about potential pitfalls and tips on how to avoid failures Presents cutting-edge material that will also interest experts in the field of bioluminescence Provides methods and assays that can be adapted and used in most specialties of biomedical and biotechnological fields: studies of cell trafficking, protein-protein interactions, intracellular signaling, apoptosis, RNAi knockdown, bacterial or viral infections, transplanted cells, stem cells proliferation, vascular flow, and tumors Covers a wide range of sub-cellular and cell-based assays for in vitro and in vivo analysis as well as non invasive, in vivo, and real time assays and monitoring for animal models Also includes methods for high throughput analysis of animal models Includes supplementary material: sn.pub/extras

Klappentext

Through the study and application of bioluminescence, scientists have painstakingly harnessed a powerful tool that enables us to seek a deeper understanding of the complex mechanisms underpinning so many vital biologic systems. In this fully revised and updated second edition of Bioluminescence: Methods and Protocols, expert researchers contribute a readable and utilitarian compilation of the newest and most innovative techniques that have emerged in this rapidly expanding and progressively diverse field including methods to assess cell trafficking, protein-protein interactions, intracellular signaling, and apoptosis. Also opening up the possibility to visualize and quantify biological mechanisms in real-time and in in vivo settings, the volume also describes the in vivo study of bacterial or viral infections, transplanted cells, stem cells proliferation, vascular flow, and tumors. Written in the highly successful Methods in Molecular Biology™ series format, chapters include brief introductions to their respective topics, lists of the necessary materials, equipment, and reagents, step-by-step, readily reproducible laboratory protocols, and notes on troubleshooting and avoiding known pitfalls.

Authoritative and cutting-edge, Bioluminescence: Methods and Protocols, Second Edition provides protocols that are detailed enough to be followed and adapted by scientific teams who have no previous expertise in bioluminescence in order to foster the potentially numerous breakthroughs and new applications from basic to applied science and medicine that must continue to be developed.

Inhalt

1. Luminescent Probes and Visualization of Bioluminescence.- 2. Validation of Bioluminescent Imaging Techniques.- 3. Assessment of Extracellular ATP Concentrations.- 4. High-Throughput Quantitative Bioluminescence Imaging for Assessing Tumor Burden.- 5. Fluorescence Imaging of Tumors with Smart pH-Activatable Targeted Probes.- 6. Imaging Vasculature and Lymphatic Flow in Mice Using Quantum Dots.- 7. Bioluminescent Imaging of Transplanted Islets.- 8. Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cell Survival, Proliferation, and Fate.- 9. Detection of Apoptosis Using Cyclic Luciferase in Living Mammals.- 10. Noninvasive Bioluminescent Imaging of Infections.- 11. Real-Time Bioluminescence Imaging of Viral Pathogenesis.- 12. Bioluminescent Monitoring of In Vivo Colonization and Clearance Dynamics by Light-Emitting Bacteria.- 13. Quantitative In Vivo Imaging of Non-viral-Mediated Gene Expression and RNAi-Mediated Knockdown.- 14. Analysis of Protein-Protein Interactions Using Bioluminescence Resonance Energy Transfer.- 15. Bioluminescent Imaging of MAPK Function with Intein-Mediated Reporter Gene Assay.- 16. Bioluminescence Analysis of Smad-Dependent TGF-? Signaling in Live Mice.- 17. Bioluminescence Imaging of Calcium Oscillations Inside Intracellular Organelles.- 18. Novel Tools for Use in Bioluminescence Resonance Energy Transfer (BRET) Assays.- 19. PIN-G Reporter for Imaging and Defining Trafficking Signals in Membrane Proteins.- 20. Imaging ?-Galactosidase Activity In Vivo Using Sequential Reporter-Enzyme Luminescence.