By Elisa Michelini, Luca Cevenini, Laura Mezzanotte, Aldo Roda (auth.), Preston B. Rich, Christelle Douillet (eds.)
Through the learn and alertness of bioluminescence, scientists have painstakingly harnessed a strong device that permits us to hunt a deeper realizing of the complicated mechanisms underpinning such a lot of very important biologic structures. during this totally revised and up to date moment version of Bioluminescence: equipment and Protocols, professional researchers give a contribution a readable and utilitarian compilation of the most recent and such a lot leading edge options that experience emerged during this speedily increasing and steadily varied box together with tips on how to verify cellphone trafficking, protein-protein interactions, intracellular signaling, and apoptosis. additionally establishing up the prospect to imagine and quantify organic mechanisms in real-time and in in vivo settings, the quantity additionally describes the in vivo examine of bacterial or viral infections, transplanted cells, stem cells proliferation, vascular circulation, and tumors. Written within the hugely winning Methods in Molecular Biology™ sequence layout, chapters comprise short introductions to their respective subject matters, lists of the mandatory fabrics, gear, and reagents, step by step, without difficulty reproducible laboratory protocols, and notes on troubleshooting and fending off identified pitfalls.
Authoritative and state of the art, Bioluminescence: tools and Protocols, moment version provides protocols which are precise sufficient to be and tailored by way of clinical groups who've no past services in bioluminescence with a purpose to foster the doubtless a number of breakthroughs and new purposes from simple to utilized technology and medication that needs to remain developed.
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Additional resources for Bioluminescence: Methods and Protocols
1998) Bioluminescence detection of ATP release mechanisms in epithelia. Am J Physiol 275, C1391–C1406. 15. , and McElroy, W. D. (1978) Purification and properties of firefly luciferase. In Methods Enzymol 57, 3–15. Chapter 4 High-Throughput Quantitative Bioluminescence Imaging for Assessing Tumor Burden Angelina Contero, Edmond Richer, Ana Gondim, and Ralph P. Mason Abstract Bioluminescence imaging (BLI) has emerged during the past 5 years as the preeminent method for rapid, cheap, facile screening of tumor growth and spread in mice.
3. 4. At the end of each assay, an ATP–luminescence relationship (calibration curve) is generated to calculate ATP concentrations. 3. Measurement of Cell Surface ATP Concentrations Using Soluble Luciferase 1. Wash the surface of cultures with PBS,3 Â . 2. 5 cm cultures. Bilaterally for polarized cultures – 1 cc and 25–500 ml to luminal and serosal side, respectively, when 12-mm Transwell is used). Equilibrate the cultures in an incubator (37°C and 5% CO2) for 1 h. 3. 2. 4. Notes 1. Firefly luciferase catalyzes the following reaction: D-luciferin þ ATP þ luciferase (L) !
Incubate for 1 h on ice. 4. Wash 3 Â with PBS. 5. 5 mg/mL purified SPA-luc (see Note 4) for 1 h at 4°C in the dark. SPA-luc will bind to the Fc domain of the antibody attached to the cells, as indicated in Step 3. 6. Wash carefully 3 Â with PBS. , HBSS/HEPES). Keep cultures in the dark at RT for 30 min to equilibrate the extracellular ATP concentrations. 2. Measurement of Cell Surface ATP Concentrations Using SPA-luc 1. Place a SPA-luc-bound cell culture in the Turner TD-20/20, add soluble luciferin (150 mM final, to the apical solution for polarized cultures) and close the lid.