Feeding behavior of flies carrying a beetle (e.g. firefly) luciferase gene can be conveniently monitored in real-time by measuring bioluminescence in 96-well microplates. Beetle luciferases catalyze a bioluminescent reaction by oxidizing D-luciferin with molecular oxygen. This also requires ATP as second substrate to first convert D-luciferin into the activated lucyferyl-adenylate form. Luciferases are widely used as reporter genes, most commonly via lytic assays of cell cultures and tissues. D-luciferin is relatively stable is solution for many days, so the beetle luciferase reaction can also be monitored in real time in live cell and tissue cultures for many days. Bioluminescence imaging is often used to track tumor development in live mice by if cancer cells are labeled with a luciferase. When injected into mice, D-luciferin is relatively quickly eliminated from the bloodstream (~30 min half-life). Drosophila flies expressing a beetle luciferase gene produce bioluminescence if fed with a food containing luciferin. Bioluminescence in flies appears within a minute and peaks within 2-3 minutes after a food ingestion. When refraining from eating or placed on a luciferin-free food, fly bioluminescence decays with about 1 hour half-life and essentially returns to the baseline after 4 hours. Naturally, flies tend to eat sporadically and often make intervals of many hours between eating. This makes beetle luciferase bioluminescence a very convenient system to monitor fly feeding timing and, to a considerable extent, food intake in real time for many days (see the Abstract Figure). In this protocol I describe one of the possible procedures to monitor fly feeding using commonly available plate readers, outline a historical background on recording bioluminescence in live flies to study circadian gene expression, illustrate several examples of feeding behaviors that can be analyzed by this procedure and discuss some potential applications. THIS PROTOCOL ACCOMPANIES THE FOLLOWING PUBLICATION Koksharov MI (2021) Monitoring fly feeding behavior and timing by beetle luciferase reporters. Protocol s.i o Protocol s.i o .
Bioluminescence and chemiluminescence are widely used in sensitive detection methods in biomedical sciences and analytical chemistry. A limitation of this type of measurements is that luminometers and platereaders do not directly quantify absolute quantum output of the reaction but report "relative luminescence units" (RLU) which are specific for a given instrument and reaction vessel design. At the same time, there are no simple and convenient luminescence reference standards that would have been universally available, so results (RLU measurements) reported by different instruments and laboratories usually cannot be directly compared.I have found that cyclodextrins -which are often used to solubilize coelenterazine (CTZ) analogs and other compounds in water buffers -catalyze a weak chemiluminescence of CTZ (and its analogs). Chemiluminescence of 20 µM CTZ in the presence of 10 mM β-cyclodextrin or 10 mM trimethyl-β-cyclodextrin in the 50 mM Naphosphate buffer (pH 7.40) can be used as a simple and convenient reference standard to define and compare RLU readings obtained by different instruments. This system is composed of only small molecules of a defined chemical composition which are not expensive and available in high purity from multiple suppliers making this system convenient for the general use as a luminescence reference standard.
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